CN115317618B - Dendrimer-coated manganese dioxide nanoparticle as well as preparation and application thereof - Google Patents

Dendrimer-coated manganese dioxide nanoparticle as well as preparation and application thereof Download PDF

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CN115317618B
CN115317618B CN202210816497.9A CN202210816497A CN115317618B CN 115317618 B CN115317618 B CN 115317618B CN 202210816497 A CN202210816497 A CN 202210816497A CN 115317618 B CN115317618 B CN 115317618B
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gppm
gox
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CN115317618A (en
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史向阳
高悦
欧阳智俊
沈思妍
贾兵洋
沈明武
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Donghua University
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Abstract

The invention relates to a dendrimer-coated manganese dioxide nanoparticle, and preparation and application thereof, wherein the dendrimer-coated manganese dioxide nanoparticle is prepared by modifying methoxy polyethylene glycol mPEG and phenylboronic acid PBA on the surface of a fifth generation polyamide-amine PAMAM dendrimer G5 and internally coating the manganese dioxide nanoparticle. The method has the advantages of simple operation process, mild reaction conditions and easy purification, and the used synthetic raw materials are environment-friendly materials, so that the method has an industrial implementation prospect.

Description

Dendrimer-coated manganese dioxide nanoparticle as well as preparation and application thereof
Technical Field
The invention belongs to the field of functional nano materials, and particularly relates to a dendrimer-coated manganese dioxide nanoparticle, and preparation and application thereof.
Background
Prevention, diagnosis and treatment of tumors have been the subject of major attention for biomedical workers worldwide. The immunotherapy can perform accurate immune killing by targeting specific tumor cells, has high specificity, low toxicity and immune memory effect, and can effectively prevent tumor recurrence and metastasis, so the immunotherapy becomes the focus of current research and gradually becomes the fourth tumor treatment means after surgery, radiotherapy and chemotherapy. However, immunosuppressive Tumor Microenvironment (TME) severely limits the therapeutic efficacy of immunotherapy. Therefore, it is important to deeply dig the mechanism of occurrence and development of tumor and develop more efficient combined immunotherapy method aiming at the characteristics of TME.
It is important that tumor cells proliferate rapidly, and the energy demand is much higher than that of normal cells, thus differing from normal cells in energy metabolism pattern. One of the biggest features is that tumor cells consume glucose far faster than normal cells and are highly dependent on glycolytic energy supply even in the presence of sufficient supply, a phenomenon also known as the "Warburg effect". Studies have shown that this phenomenon limits the function of immune cells in TMEs.
Among the numerous nanocarrier materials, polyamide-amine dendrimers (poly (amidoamine), PAMAM) are of great interest in the nanocarrier field due to their unique physicochemical properties. It is a highly branched, synthetic, highly monodisperse macromolecule with very precise core, internal space and a large number of surface functional groups. The PAMAM dendrimer has good carrier performance after being functionalized by the surface functional groups, and can target and deliver anticancer drugs, genes and the like to tumor sites.
According to the search results of domestic and foreign documents, no related report on the preparation of dendrimer-coated manganese dioxide nanoparticles and the application of the dendrimer-coated manganese dioxide nanoparticles in tumor drug delivery and combined treatment is found at present.
Disclosure of Invention
The invention aims to provide a dendrimer-coated manganese dioxide nanoparticle, and preparation and application thereof.
The invention relates to a dendrimer composite material, which is prepared by modifying methoxy polyethylene glycol mPEG and phenylboronic acid PBA on the surface of a fifth generation polyamide-amine PAMAM dendrimer G5 and internally wrapping manganese dioxide nano particles.
The invention relates to a preparation method of a dendrimer composite material, which comprises the following steps:
(1) Mixing an aqueous solution of mPEG (Mal-mPEG) with a maleimide terminal group and an aqueous solution of a fifth-generation polyamide-amine PAMAM dendrimer G5, stirring in a water bath, dialyzing, purifying, and freeze-drying to obtain the fifth-generation polyamide-amine PAMAM dendrimer G5-mPEG modified with methoxy polyethylene glycol mPEG;
(2) Mixing the G5-mPEG solution and the 4-bromomethyl phenylboronic acid solution, stirring in a water bath for reaction, dialyzing, purifying and freeze-drying to obtain a fifth generation polyamide-amine PAMAM dendrimer G5-mPEG-PBA modified with phenylboronic acid molecules, which is called GPP for short;
(3) Adding KMnO 4 aqueous solution into G5-mPEG-PBA aqueous solution, stirring for reaction, and dialyzing
Purifying and freeze-drying to obtain the dendrimer composite material G5-mPEG-PBA@MnO 2, which is simply referred to as GPPM. The preferred mode of the preparation method is as follows:
The molar ratio of the fifth generation polyamide-amine PAMAM dendrimer G5 to the Mal-mPEG in the step (1) is 1:12-1:15, and the water bath stirring is carried out for 24-36h under the condition of 28-32 ℃.
The solvent of the solution in the step (2) is dimethyl sulfoxide (DMSO), the molar ratio of the G5-mPEG to the 4-bromomethyl phenylboronic acid is 1:50-1:55, and the water bath stirring reaction is carried out for 24-36h at 68-72 ℃.
The KMnO 4 aqueous solution is added in the step (3), specifically, KMnO 4 aqueous solution is added at a constant speed through a micro-injection pump, and the flow rate of the micro-injection pump is 0.8-1.0 mL/min.
The molar ratio of the G5-mPEG-PBA to the KMnO 4 in the step (3) is 1:30-55, the concentration of the KMnO 4 solution is 0.25-0.30 mg/mL, and the stirring reaction is carried out at room temperature for 1-1.5h.
Further preferably, the molar ratio of the G5-mPEG-PBA to the KMnO 4 is 1:50-55.
And (3) dialyzing in the steps (1) - (3) by using a cellulose dialysis membrane with the molecular weight cut-off of 10000 in ultrapure water for 1-3 days. The invention provides a dendrimer composite material prepared by the method.
It is further preferred that the dialysis time in steps (1) - (2) is 2-3 days, and that the dialysis time in step (3) is 1 day. The invention relates to a drug-loaded dendrimer composite material, which is prepared by compounding glucose oxidase GOx and/or an interferon gene stimulating factor STING agonist cGAMP with a carrier.
The invention relates to an application of the drug-loaded dendrimer composite material in preparing a chemical kinetics/hunger/immunity combined therapeutic drug for tumors.
The invention provides an application of dendrimer-coated manganese dioxide nanoparticles as a carrier system in tumor nano-drugs, which can be used for combining glucose oxidase (GOx) and an interferon gene stimulating factor (STING) agonist cGAMP for chemical kinetics/hunger/immune combination treatment of tumors.
The method comprises (1) a preparation method of a functionalized dendrimer G5-mPEG-PBA (GPP for short), (2) a preparation method of a dendrimer-coated manganese dioxide nanoparticle G5-mPEG-PBA@MnO 2 (GPPM for short), and (3) application of a GPPM nanoparticle composite GOx and a STING agonist cGAMP as nano medicines in combined treatment of tumors.
The invention is based on fifth generation polyamide-amine dendrimer, surface modification mPEG and PBA, and construction of GPPM nanometer particles by internally wrapping manganese dioxide. Experimental results show that GPPM nano particles have excellent stability, chemical kinetics performance and carrier performance, can be used as a carrier composite GOx and cGAMP to construct a delivery system, can realize good combined tumor treatment effect in vitro and in vivo, and can particularly and simultaneously effectively inhibit the growth of primary tumors and remote tumors in mice.
The method comprises the steps of characterizing GPPM nano particles prepared by using nuclear magnetic resonance hydrogen spectrum (1 H NMR), dynamic Light Scattering (DLS) test, zeta potential test, transmission Electron Microscope (TEM), X-ray photoelectron spectroscopy (XPS) analysis, relaxation rate test and the like, evaluating chemical kinetics performance of materials in vitro through Methylene Blue (MB) degradation experiments, verifying cancer cell killing effect of GPPM loaded with GOx in vitro through CCK-8 method, detecting expression condition of ROS and Lipid Peroxide (LPO) through probes, measuring consumption condition of intracellular Glutathione (GSH) through a kit, evaluating chemical kinetics treatment effect of GPPM loaded with GOx through the kit, evaluating cancer cell apoptosis caused by a delivery system through GPPM co-loaded GOx and cGAMP, evaluating tumor cell immunogenic death effect caused by the delivery system through Transwell experiments, evaluating maturation condition of antigen presenting cell Dendritic Cells (DC), and finally constructing primary tumor and distal tumor in vivo and evaluating in vivo combined injection effect of the tumor. The specific test results are as follows:
(1) 1 H NMR characterization results
The analysis result of the nuclear magnetic resonance hydrogen spectrum is shown in fig. 2, wherein fig. 2a is a graph of G5-mPEG, wherein 2.2-3.4 ppm is a characteristic peak of methylene protons in G5, and 3.6ppm is a characteristic peak of mPEG, and 10.0 mPEG molecules connected to each G5 can be calculated through integration. FIG. 2b shows the nuclear magnetic resonance hydrogen spectrum of GPP materials, and characteristic peaks of PBA molecules at 7.0-8.0ppm are attributed, which indicates successful connection of PBA molecules, and the total number of PBAs connected on each G5 is 27.5 through integral calculation.
(2) Optimization experiment of manganese dioxide wrapping metering ratio
The molar ratio of GPP to KMnO 4 is set to be 1:30, 1:50 or 1:80 for wrapping manganese dioxide, the obtained product solution is shown in figure 3, wherein the molar ratio of 1:30 to 1:50 is selected to obtain stable dendrimer-wrapped manganese dioxide nano particles, the product solution is clear and transparent, and the molar ratio of 1:80 is selected to generate particle precipitation, so that stable products cannot be obtained. The invention adopts the optimized ratio of 1:50 to synthesize the G5-mPEG-PBA@MnO 2 (namely GPPM).
(3) Hydrodynamic diameter and Zeta potential test
The synthesized GPP and GPPM were dissolved in ultrapure water, and Zeta potential and DLS tests were performed, and the results are shown in Table one. It was found that GPPM has a suitable nanoscale, and that GPPM potential is reduced compared with GPP due to encapsulation of manganese dioxide, probably because the surface amino groups of dendrimers participate in the reduction reaction of KMnO 4, thereby reducing the surface density, and that hydrodynamic diameter is reduced, probably because the dispersibility is improved, thereby further reducing the tendency of the dendrimers to aggregate.
(4) TEM test
The morphology of the inner core MnO 2 is observed by taking GPPM nano particles for TEM test, and the result is shown in figures 4a-b, and the monodispersity of the GPPM inner core MnO 2 nano particles can be found to be good, wherein the particle size of the wrapped manganese dioxide nano particles is relatively uniform. As shown in fig. 4c, the average size of manganese dioxide nanoparticles was 2.8nm as indicated by particle size statistics.
(5) XPS analysis
The GPPM nano-particles are weighed for XPS test, the test result is shown in figure 5, peaks of Mn2p 3/2 and Mn2p 1/2 spin orbitals of Mn element appear in a spectrogram, the valence state of Mn element in GPPM synthesized is positive tetravalent, and the successful synthesis of manganese dioxide is indicated.
(6) T 1 relaxation behavior characterization
Manganese dioxide is capable of consuming GSH present in excess in TME, yielding Mn 2+ with good T 1 imaging properties, and therefore the r 1 relaxation rate of GPPM after GSH treatment was next studied. The Mn content of GPPM nanoparticles was first determined by ICP-OES analysis, followed by two sets of GPPM solutions of different Mn concentrations (0.125, 0.25, 0.5, 1.0 and 1.5 mM) were prepared, with 10mM GSH added to one set of solutions for pretreatment. The relaxation properties of the two groups of solutions were characterized by T 1 respectively, and comparison shows that after GSH treatment, the relaxation rate of Mn 2+ and GPPM is as high as 8.37mM -1s-1, and MnO 2 in GPPM can exist stably in the absence of GSH, so that the relaxation rate is only 0.17mM -1s-1. The test results indicate that the synthesized GPPM nanoparticles have the potential to perform T 1 MR imaging after GSH treatment.
(7) MB degradation experiment
Mn 2+ is capable of generating Fenton-like reaction with H 2O2 in TME in the presence of HCO 3 - in physiological environment to generate cell killing hydroxyl radicals, while GSH inhibits the generation of hydroxyl radicals. Therefore, on the basis of MB degradation in an oxidation environment, a colorimetry is adopted to detect GPPM the capability of generating hydroxyl radicals in GSH environments with different concentrations. NaHCO 3/CO2 buffer ([ NaHCO 3/CO2 ] =25 mM) containing GSH (0, 1,2, 5 or 10 mM), GPPM nano particles ([ Mn 2+]＝0.25mM)、H2O2 (10 mM) and MB (10 μg/mL) at different concentrations was prepared, each set of solutions was left to stand in a 37 ℃ environment for 30 minutes, and after the reaction was completed, a photograph was taken and an ultraviolet absorption curve was measured.
(8) CCK-8 cytotoxicity assay
GPPM and GOx were dissolved in PBS solution and incubated for 30 min with mixing at a certain ratio, and GPPM complexing with GOx (GPPM@GOx) was performed. Subsequently, the cancer cell killing effect of GOx, GPPM and GPPM@GOx was initially studied by CCK-8 cytotoxicity experiments using CT26 mouse colorectal cancer cells as a model. Cytotoxicity tests (0, 5, 10, 20, 50 and 100 ng/mL) were first performed for different concentrations of GOx, which showed good cancer cell killing effect as shown in fig. 8a, with CT26 cell viability of only 26.5% at 100 ng/mL. The concentrations of gox=50 ng/mL were then selected for the composite test of cytotoxicity with GPPM (0, 2, 5, 10, 20 and 50 μg/mL) at different concentrations, as shown in fig. 8b, GPPM was able to effectively reduce cancer cell viability with increasing Mn concentration, whereas cytotoxicity was significantly enhanced after the composite GOx. This is probably due to the ability of GOx to catalyze the glucose production H 2O2, further enhancing the Fenton-like reaction mediated by GPPM upon release of manganese ions.
(9) In vitro pharmacokinetic treatment evaluation.
A series of evaluations of the chemokinetic therapeutic effects of GPPM and GPPM@GOx were performed with GOx concentrations of 50ng/mL and Mn concentrations of 10 μg/mL, including the effect of the material on the production of ROS, LPO by cancer cells and the ability to consume intracellular GSH. In the experimental process, to facilitate evaluation of the effect of GOx complex, a complex of GPPM and Bovine Serum Albumin (BSA) without special function at the same concentration was set as an experimental control group. As shown in FIGS. 9a-b and 10, the effect of ROS and LPO production by cancer cells treated with different materials was first examined by flow cytometry and confocal laser microscopy, respectively, using ROS probe (DCFH-DA) and LPO probe (C11-BODIPY). Experimental results show that GPPM has good intracellular chemical kinetics reaction performance, the effect of the compound GOx is further enhanced, the generation of ROS and the expression of LPO in cells can be mediated efficiently, and the compound BSA has no enhancement effect. As shown in fig. 11, further using GSH kit to detect intracellular GSH levels in cancer cells of different experimental groups, the results obtained showed that both GPPM and gppm@gox complexes were very effective in scavenging intracellular GSH, the gppm@gox complex was better, further proving the good chemokinetic therapeutic effects of GPPM and gppm@gox complexes.
(10) Apoptosis experiments
GPPM, GOx and cGAMP prepared by the method are dissolved in PBS, mixed in proportion and incubated for 30 minutes, meanwhile, the GOx and the cGAMP are compounded (Mn=10 mug/mL, GOx=50 ng/mL, cGAMP=1 mug/mL) and then subjected to apoptosis experimental evaluation, and five experimental groups of PBS, GOx, GPPM, GPPM@GOx and GPPM@GOx+cGAMP are arranged together and detected by adopting an Annexin V-FITC/PI method. As shown in FIGS. 12a-b, GPPM can effectively cause chemokinetic cell killing compared with PBS group and GOx group, so that more cells can be apoptosis and necrosis are produced, GPPM@GOx and GPPM@GOx+cGAMP experimental groups can effectively cause massive apoptosis and necrosis of cancer cells due to the combined action of GPPM and GOx, and it is noted that the compound cGAMP in the GPPM@GOx+cGAMP experimental groups can be beneficial to apoptosis of tumor cells through activation of TBK1-IRF3 signal channels, so that the optimal cancer cell apoptosis and necrosis effects are obtained.
(11) Evaluation of in vitro immunogenic death (ICD) Effect
The release of the immunogenic death markers HMGB1 and ATP from CT26 cancer cells treated with different experimental group materials (mn=10 μg/mL, gox=50 ng/mL, cgamp=1 μg/mL) was examined, and the effect of inducing immunogenic death of cancer cells in vitro after GPPM combinations of GOx and cGAMP was evaluated. As shown in fig. 13 and 14, gppm@gox+cgamp, in which GOx and cGAMP are combined, is most effective in inducing ICD production by tumor cells, in favor of activating immune response, compared to other experimental groups, due to the presence of chemokinetic killing action of the vector itself, starvation treatment of the enzyme, and activation of TBK1-IRF3 signaling pathway by cGAMP.
(12) Evaluation of immune Effect in vitro
The expression of the surface maturation markers CD80 and CD86 of DCs was detected by flow cytometry 24 hours later on the lower co-cultured DCs using Transwell experiments, the upper layer was incubated with CT26 cancer cells and treated with different experimental group materials (mn=10 μg/mL, gox=50 ng/mL, cgamp=1 μg/mL) and the ability of the materials to activate immune responses in vitro was evaluated. As shown in fig. 15a-b, the gppm@gox+cgamp experimental group has the highest maturation degree compared with other experimental groups, and the gppm@gox+cgamp experimental group DC is favorable for the progress of anti-tumor immune response, since the gppm@gox+cgamp experimental group can most remarkably trigger cancer cells to generate ICD, and the composite cGAMP can directly activate STING in DC, promote the generation of type I IFN and inflammatory cytokines, and initiate an innate immune response.
(13) Evaluation of in vivo antitumor Effect
As shown in fig. 16, BALB/C mice were randomly grouped (n=5), 1×10 6 CT26 cells were subcutaneously planted in the right leg to construct a primary subcutaneous tumor model of the mice, and when the tumor volume was about 50mm 3 (after 8 days), treatment was performed on each of days 1, 5 and 9, i.e., PBS, GOx, cGAMP, GPPM, gppm@gox or gppm@gox+cgamp ([ cGAMP ] =5 μg/time), respectively, and the tumor volume, weight, etc. of the mice were monitored for 21 consecutive days. Meanwhile, in order to evaluate the in vivo anti-tumor immune activation of the treatment group, 5×10 5 CT26 cells were planted subcutaneously in the left leg of the mice at the time of the first treatment to construct a distal tumor, and the growth of the distal tumor was monitored for 21 consecutive days as well.
As shown in fig. 17, the mice of each group had a slow increase in body weight over time, and there was no significant difference between each group, indicating that there was no significant systemic toxicity after the material injection of each experimental group. In the case of primary tumor growth, as shown in fig. 18, the primary tumors of mice in the PBS group, the GOx group and the cGAMP group all grow rapidly, the tumor growth of mice in the GPPM experimental group is inhibited to a certain extent, the tumor growth of the mice in the GPPM@GOx and the GPPM@GOx+cgamp experimental group is obviously inhibited, wherein the tumor inhibition effect of the GPPM@GOx+cgamp experimental group is the best, the tumor of the mice in the experimental group is almost completely resolved, and the tumor inhibition rate is as high as 99.3%. In the case of the growth of the distal tumor, as shown in fig. 19, the growth of the distal tumor in the mice in the PBS group is also rapid, the growth rate of the distal tumor in the GOx group and the cGAMP group is slower than that in the PBS group, the GPPM group also has a better effect of inhibiting the distal tumor due to the stimulation of the innate immunity by Mn 2+, the gppm@gox group combines Mn 2+ with GOx, the effect of inhibiting the distal tumor is better, and simultaneously, the growth of the distal tumor in the mice is completely inhibited by implementing the chemical kinetics/starvation/immunity combined treatment in the gppm@gox+cgamp experimental group. More intuitively, as shown in fig. 20a-b, it can be seen from the representative dig map of the mouse tumor the last day of the experiment that the gppm@gox+cgamp experimental group had the best tumor suppression effect, only one mouse remained with a small primary tumor, while no mouse developed a distant tumor.
The effect of different sets of materials to elicit an immune response in vivo was further evaluated. After the experiment is finished, the spleen of the mouse is planed, T cells are obtained through separation by a nylon column method, and flow analysis of CD4 and CD8+ T cells is carried out, as shown in figure 21, the highest differentiation degree of CD8+ killer T cells in the spleen of the mouse of the GPPM@GOx+cGAMP experiment group can be found, and the highest anti-tumor immune response degree is shown. Meanwhile, FOXP3, CD25 and CD4 are selected as markers, and expression conditions of immune suppression type regulatory T cells (Tregs, cd4+cd25+foxp3+) are analyzed through a flow, as shown in fig. 22, the mice of the gppm@gox+cgamp experimental group have the least population proportion of Tregs cells, so that immune killing of organisms to tumors is facilitated, and the best anti-tumor immune response effect of the mice of the gppm@gox+cgamp experimental group is further proved.
Advantageous effects
(1) The method has the advantages of simple operation process, mild reaction conditions and easy purification, and the used synthetic raw materials are environment-friendly materials, so that the method has an industrialized implementation prospect;
(2) The dendrimer-coated manganese dioxide nanoparticle prepared by the invention has good stability, chemical kinetics performance and carrier performance, and can be used for preparing tumor nano-drugs by compounding GOx and cGAMP;
(3) The dendrimer prepared by the invention can be used for the high-efficiency combined treatment of in-vitro and in-vivo tumor cells after wrapping manganese dioxide nanoparticle composite GOx and cGAMP, wherein the growth of primary tumors and remote tumors can be inhibited in vivo at the same time, and the in-vivo anti-tumor immune response can be effectively stimulated;
(4) The dendrimer-coated manganese dioxide nanoparticle prepared by the invention has potential application value in the fields of nano medicine, tumor combined treatment and diagnosis and treatment integration.
Drawings
FIG. 1 is a schematic diagram of the synthesis process of dendrimer-coated manganese dioxide nanoparticles according to the present invention.
FIG. 2 is 1 H NMR spectra of G5-mPEG (a) and GPP (b) prepared according to the present invention.
FIG. 3 is a photograph of a product solution of GPP prepared according to the present invention coated with manganese dioxide at various dendrimer/KMnO 4 molar ratios.
FIG. 4 is a high resolution TEM image (a-b) of GPPM nanoparticles prepared according to the present invention and corresponding (c) particle size distribution histogram of manganese dioxide.
FIG. 5 is a Mn 2P XPS spectrum of GPPM nanoparticles prepared according to the present invention.
FIG. 6 is a graph of the r 1 relaxation rate results of GPPM nanoparticles prepared according to the present invention with or without GSH pretreatment.
FIG. 7 is a photograph of GPPM nanoparticles prepared according to the present invention mixed with MB, H 2O2 and GSH at various concentrations in NaHCO 3 buffer and UV absorption curve (the mixture was placed in 37 ℃ C. For 30min after preparation, and [ NaHCO 3/CO2]＝25mM,[MB]＝10μg/mL,[H2O2 ] = 10mM, [ Mn ] = 0.25 mM).
Fig. 8 shows the toxicity of GOx to CT26 cells at different concentrations (a) and the toxicity of GPPM and gppm@gox complex at different Mn concentrations (gox=50 ng/mL) to CT26 cells (b).
FIG. 9 is a graph of the ROS-flow fluorescence profile (a) and quantitative results (b) of CT26 cells after 4 hours of treatment with different materials. Fig. 10 is a confocal laser photograph of LPO expression in CT26 cells (nuclei stained with DAPI) after 4 hours of treatment with different materials.
FIG. 11 is a graph showing the results of detection of intracellular GSH levels in CT26 after 4 hours of treatment with different materials.
FIG. 12 is a plot (a) of the flow assay for apoptosis of CT26 cells and a plot (b) of the quantitative ratio of necrotic/apoptotic cells after 24 hours of treatment with different materials.
FIG. 13 is a graph showing the results of measuring the amount of HMGB1 released from CT26 cells after 24 hours of treatment with different materials.
FIG. 14 is a graph showing the results of the measurement of ATP release from CT26 cells after 24 hours of treatment with different materials.
FIG. 15 is a graph (a) showing the flow assay profile and quantitative results (b) of the surface maturation markers CD80 and CD86 of DC after 24 hours of co-incubation with CT26 cells treated with different materials.
FIG. 16 is a schematic of the experimental procedure of in vivo anti-tumor in mice of the present invention.
Fig. 17 is a graph of body weight change in CT26 tumor-bearing mice over 21 days after receiving PBS, GOx, cGAMP, GPPM, gppm@gox or gppm@gox+cgamp treatment.
Fig. 18 is a plot of primary tumor volume change in CT26 tumor-bearing mice over 21 days after receiving PBS, GOx, cGAMP, GPPM, gppm@gox or gppm@gox+cgamp treatment.
Fig. 19 is a graph of distal tumor volume change in CT26 tumor-bearing mice over 21 days after receiving PBS, GOx, cGAMP, GPPM, gppm@gox or gppm@gox+cgamp treatment.
FIG. 20 is a photograph of primary tumor (a) and distant tumor (b) of different further experimental groups of mice after the end of the 21 day in vivo experimental period (1: PBS;2: GOx;3: cGAMP;4: GPPM;5: GPPM@GOx;6: GPPM@GOx+cGAMP).
FIG. 21 is a flow assay of CD4+/CD8+ T cells in spleens of mice from different further groups after the end of the 21 day in vivo experimental period.
Fig. 22 shows the results of flow-through detection of Tregs in spleens of mice from different further groups after the end of the 21-day in vivo experimental period.
Detailed Description
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Example 1
(1) 46Mg of Mal-mPEG (available from Shanghai inflammatory Yi Bio Inc.) and 50mg of fifth generation PAMAM dendrimer (G5) (available from Dendritech Co., U.S.A.) were weighed and dissolved in ultrapure water, and the Mal-mPEG solution was added to the G5 solution, followed by stirring reaction in a water bath at 30℃for 24 hours. After the reaction is finished, dialyzing in pure water for 3 days by using a cellulose dialysis bag with the molecular weight cut-off of 10000, and freeze-drying to obtain G5-mPEG powder;
(2) 50mg of G5-mPEG powder was weighed and dissolved in DMSO solution, 11.7mg of 4-bromomethylbenzoic acid in DMSO solution (available from Shanghai Ala Biotechnology Co., ltd.) was added, and the reaction was stirred in a water bath at 70℃for 24 hours. After the reaction, dialyzing in pure water for 3 days by using a cellulose dialysis bag with the molecular weight cut-off of 10000, and freeze-drying to obtain the GPP material.
(3) Weighing 50mg of GPP, dissolving in ultrapure water, adding 0.25mg/mL of KMnO 4 aqueous solution at a constant speed (the flow rate is 0.8-1.0 mL/min) by a microinjection pump according to the mol ratio of GPP to KMnO 4 of 1:50, and stirring for reaction for 1 hour after the dripping is finished. After the reaction is finished, dialyzing in pure water for 1 day by using a cellulose dialysis bag with the molecular weight cutoff of 10000, and then freeze-drying to obtain dendrimer-coated manganese dioxide nanoparticles, namely GPPM nanoparticles.
Example 2
Characterization of GPPM nanoparticles prepared in example 1 and related intermediates in the preparation process:
(1) The G5-mPEG is weighed and dissolved in 600 mu L of deuterated water for nuclear magnetic hydrogen spectrum analysis, the analysis result is shown in figure 2a, wherein 2.2-3.4 ppm is the characteristic peak of methylene protons in G5, and 3.6ppm is the characteristic peak of mPEG, and 10.0 mPEG molecules connected to each G5 can be calculated through integration. The GPP was also weighed and dissolved in 600. Mu.L of deuterated water for nuclear magnetic hydrogen spectrum analysis, and the analysis results are shown in FIG. 2b, and characteristic peaks of PBA molecules at 7.0-8.0ppm are attributed, which indicates successful connection of PBA, and the total number of PBA connected on each G5 is 27.5 through integral calculation.
(2) The molar ratio of GPP to KMnO 4 is set to be 1:30, 1:50 or 1:80 for wrapping manganese dioxide, the obtained product solution is shown in figure 3, wherein the molar ratio of 1:30 to 1:50 is selected to obtain stable dendrimer-wrapped manganese dioxide nano particles, the product solution is clear and transparent, and the molar ratio of 1:80 is selected to generate particle precipitation, so that stable products cannot be obtained. Thus, GPPM synthesis was optimally performed using a 1:50 ratio. The synthesized GPP and GPPM were dissolved in ultra pure water, and Zeta potential and DLS diameter tests were performed, and GPPM nanoparticles were found to have a smaller nanoscale than GPP, while GPP was at 19.2mV potential, while GPPM potential was reduced to 9.4mV due to inclusion of manganese dioxide.
In order to characterize the morphology of GPPM materials, GPPM solution was dropped on a copper mesh, and after sufficient drying, TEM testing was performed, and the obtained results are shown in fig. 4a-b, and it can be found that the monodispersity of GPPM nanoparticles is good, wherein the particle size of the encapsulated manganese dioxide nanoparticles is relatively uniform. As shown in FIG. 4c, the average size of the manganese dioxide nanoparticles was 2.8nm as indicated by the particle size statistics. And weighing GPPM nano particles for XPS test to analyze the valence state of the Mn element, wherein the test result is shown in figure 5, peaks of Mn2p 3/2 and Mn2p 1/2 spin orbits of the Mn element appear in a spectrogram, the valence state of the Mn element in the synthesized GPPM is positive tetravalent, and the successful synthesis of manganese dioxide is indicated.
Table 1GPP and GPPM potential particle size test results for materials
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
Example 3
Material T 1 imaging relaxation performance test was performed on GPPM prepared in example 1.
Manganese dioxide is able to consume GSH present in excess in TME, yielding Mn 2+ with good T 1 imaging properties, and thus the r 1 relaxation rate of GPPM after GSH treatment was subsequently investigated. The Mn content of GPPM nanoparticles was first determined by ICP-OES analysis, followed by two sets of GPPM solutions of different Mn concentrations (0.125, 0.25, 0.5, 1.0 and 1.5 mM) were prepared, with 10mM GSH added to one set of solutions for pretreatment. The relaxation properties of the two groups of solutions were characterized by T 1 respectively, and comparison shows that after GSH treatment, the relaxation rate of Mn 2+ and GPPM is as high as 8.37mM -1s-1, and MnO 2 in GPPM exists stably in the absence of GSH, so that the relaxation rate is only 0.17mM -1s-1. The test results indicate that the synthesized GPPM nanoparticles have the potential to perform T 1 MR imaging after GSH treatment.
Example 4
GPPM prepared in example 1 was tested for its ability to generate hydroxyl radicals in vitro.
Mn 2+ is capable of generating Fenton-like reaction with H 2O2 in TME in the presence of HCO 3 - in physiological environment to generate cell killing hydroxyl radicals, while GSH inhibits the generation of hydroxyl radicals. Therefore, on the basis of MB degradation in an oxidation environment, a colorimetry is adopted to detect GPPM the capability of generating hydroxyl radicals in GSH environments with different concentrations. NaHCO 3/CO2 buffer ([ NaHCO 3/CO2 ] =25 mM) containing GSH (0, 1,2, 5 or 10 mM), GPPM nano particles ([ Mn 2+]＝0.25mM)、H2O2 (10 mM) and MB (10 μg/mL) at different concentrations was prepared, each set of solutions was left to stand in a 37 ℃ environment for 30 minutes, and after the reaction was completed, a photograph was taken and an ultraviolet absorption curve was measured.
Example 5
Cytotoxicity after GPPM was compounded with GOx prepared in example 1 was tested. GPPM and GOx are dissolved in PBS solution, mixed and incubated for 30 minutes according to a certain proportion, the composition of GPPM and GOx (GPPM@GOx) is carried out, CCK-8 cytotoxicity experiment is carried out by taking CT26 mouse colorectal cancer cells as a model, and the cancer cell killing effect of GOx, GPPM and GPPM@GOx is primarily studied.
First, the cytotoxicity of GOx alone was studied, CT26 cells were seeded in 96-well plates at 8000/well density, cultured for 12 hours, and after cell attachment, the medium was replaced with fresh medium containing GOx at different concentrations (0, 5, 10, 20, 50 and 100 ng/mL). After further culturing for 24 hours, the medium was replaced with a serum-free medium containing 10% CCK-8 solution (purchased from Shanghai Biyun biotechnology Co., ltd.) and cultured in a 37℃incubator for 2 hours, and then the absorbance was measured at 450nm by using an enzyme-labeled instrument to calculate the cell viability. As shown in fig. 8a, GOx showed good cancer cell killing effect with CT26 cell viability of only 26.5% at 100 ng/mL.
Similarly, CCK-8 cytotoxicity assays of GPPM and GPPM@GOx were performed. As shown in FIG. 8b, GPPM can effectively reduce the activity of cancer cells with the increase of Mn concentration, and the cytotoxicity is obviously enhanced after compounding GOx, by selecting Mn element concentrations of 0, 2,5, 10, 20 and 50 mug/mL and GOx concentration of 50ng/mL for experiments. This is probably due to the ability of GOx to catalyze the glucose production H 2O2, further enhancing the Fenton-like reaction mediated by GPPM upon release of manganese ions.
Example 6
The effect of the GPPM prepared in example 1 on the in vitro chemical kinetics treatment after compounding GOx was tested. A series of evaluations of the chemokinetic therapeutic effects of GPPM and GPPM@GOx were performed with GOx concentrations of 50ng/mL and Mn concentrations of 10 μg/mL, including the effect of the material on the production of ROS, LPO by cancer cells and the ability to consume intracellular GSH. In the experimental process, to facilitate evaluation of the effect of GOx complex, a complex of GPPM and BSA without special function at the same concentration was set as an experimental control group.
Firstly, detecting ROS, planting CT26 cells in a 12-well plate at the density of 1X 10 5/well, changing fresh culture medium after the cells are attached, respectively adding the culture medium containing PBS, GOx, GPPM, GPPM@BSA or GPPM@GOx for incubation for 4 hours, and washing with PBS three times after the culture is finished. Subsequently, a solution (1 mL) of DCFH-DA detection probe (purchased from Shanghai Biyun Biotechnology Co., ltd.) diluted 500-fold with serum-free RPMI 1640 medium was added under light-shielding conditions, and after incubation for 20 minutes, cells were washed, digested and collected by centrifugation for flow cytometry detection. As shown in fig. 9a-b, the experimental results show that GPPM has good intracellular chemical kinetics reaction performance, and better effect after compounding GOx, the ROS fluorescent signal of the cell is obviously enhanced, and no enhancement effect is obtained after compounding BSA.
The intracellular LPO expression level was then further observed using a laser confocal microscope. CT26 cells were seeded at a density of 1X 10 5/well in a laser confocal microscope dish at 37℃overnight in a 5% CO 2 incubator, fresh medium was changed after cell attachment, medium containing PBS, GOx, GPPM, GPPM@BSA or GPPM@GOx was added separately for incubation for 4 hours, and after incubation was completed, washed three times with PBS. Under dark conditions, 1 μ L C-BODIPY probe (available from Shanghai Biyun Biotechnology Co.) and 1mL PBS were added to each well, after 20min incubation, washed three times with PBS and fixed with 2.5% glutaraldehyde for 15 min, after fixation stained with DAPI for 10 min, and then fluorescent signals were observed under a confocal microscope oil. As shown in fig. 10, the oxidation state fluorescence signal was strongest and the non-oxidation state fluorescence signal was weakest for gppm@gox group cells, indicating that LPO expression was greatest for this experimental group of cells.
Intracellular GSH levels were further detected in cancer cells from different experimental groups using GSH kits. CT26 cells were planted in 12-well plates at a density of 1X 10 5/well, after the cells were attached, fresh medium was changed, medium containing PBS, GOx, GPPM, GPPM@BSA or GPPM@GOx was added to each for 4 hours, and after the culture was completed, the cells were collected by washing with PBS three times, digestion and centrifugation. The cells were then sonicated and centrifuged to extract the cell sap, and GSH content was tested in the operating procedure using GSH testing kit (purchased from the institute of bioengineering, built in south kyo). As shown in fig. 11, the results obtained show that both GPPM and gppm@gox complexes are very effective in scavenging intracellular GSH, the gppm@gox complex is better, further proving the good chemical kinetics of both GPPM and gppm@gox complexes.
Example 7
The GPPM composite GOx and cGAMP prepared in example 1 was tested for its effect of inducing apoptosis in cancer cells. The prepared GPPM, GOx and cGAMP were dissolved in PBS, mixed in proportion, and incubated for 30 minutes for complexation, followed by experimental evaluation.
CT26 cells were seeded at a density of 2×10 5/well in 6-well plates, after cell attachment, fresh medium was changed, 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, incubated for 24 hours, after the end of the incubation, the cells were washed three times with PBS, digested and centrifuged, and collected, 500 μl of buffer working solution, 5 μl of Annexin V-FITC staining and 5 μl of PI staining solution (reagents were all purchased from Jiangsu key biotechnology co) were sequentially added, followed by detection with a flow cytometer. As shown in FIGS. 12a-b, GPPM can effectively cause chemokinetic cell killing compared with PBS group and GOx group, so that more cells can be apoptosis and necrosis are produced, GPPM@GOx and GPPM@GOx+cGAMP experimental groups can effectively cause massive apoptosis and necrosis of cancer cells due to the combined action of GPPM and GOx, and it is noted that the compound cGAMP in the GPPM@GOx+cGAMP experimental groups can be beneficial to apoptosis of tumor cells through activation of TBK1-IRF3 signal channels, so that the optimal cancer cell apoptosis and necrosis effects are obtained.
Example 8
The GPPM prepared in example 1 was tested for its ability to produce ICD effects in vitro after complexing GOx and cGAMP.
The release of the immunogenic death markers HMGB1 and ATP was first examined 24 hours after CT26 cancer cells were treated with different experimental group materials. CT26 cells were seeded at a density of 2×10 5/well in 6-well plates, after cell attachment, fresh medium was changed, 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, and after the end of the incubation, cell culture supernatants were collected and subsequently tested using HMGB1 assay kit (available from wunschel biology ltd) and ATP assay kit (available from shanghai bi-cloudy biotechnology ltd), respectively. As shown in fig. 13 and 14, gppm@gox+cgamp, in which GOx and cGAMP are combined, is most effective in inducing ICD production by cancer cells, in favor of activating immune response, compared to other experimental groups, due to the presence of chemokinetic killing action of the vector itself, starvation treatment of enzymes, and activation of TBK1-IRF3 signaling pathway by cGAMP.
Example 9
Test example 1 in vitro immune effects after GPPM complex GOx and cGAMP were prepared.
CT26 cells were first seeded at a density of 5X 10 4/well in the upper chamber of a Transwell plate (12-well plate, cell aperture 3 μm) and DC were first seeded at a density of 1X 10 5/well in the lower chamber. After cell attachment, the medium in the upper chamber was replaced with fresh PBS, GOx, GPPM, gppm@gox or gppm@gox+cgamp-containing medium (mn=10 μg/mL, gox=50 ng/mL, cgamp=1 μg/mL) and transferred to the lower layer for co-cultivation for 24 hours. Following digestion and centrifugation, DCs were collected, stained with fluorescently labeled CD80 and CD86 antibodies, and the ability of the material to activate immune responses in vitro was assessed by detecting expression of DC maturation markers using a flow cytometer. As shown in fig. 15, the gppm@gox+cgamp experimental group has the highest maturation degree compared with the other experimental groups, and the gppm@gox+cgamp experimental group DC has the highest maturation degree, so that the anti-tumor immune response is facilitated, because the gppm@gox+cgamp experimental group can most remarkably trigger cancer cells to generate ICD, and the composite cGAMP can directly activate STING in DC, promote the generation of type I IFN and inflammatory cytokines, and initiate an innate immune response.
Example 10
The in vivo antitumor effect of GPPM in example 1 after compounding GOx and cGAMP was evaluated.
Female BALB/C mice of 5 weeks of age used in the experiments were purchased from Shanghai Laek's laboratory animal center. As shown in fig. 16, BALB/C mice were randomly grouped (n=5), 1×10 6 CT26 cells were subcutaneously planted in the right leg to construct a primary tumor of the mice, and the tumor volume, weight, etc. of the mice were monitored for 21 consecutive days after treatment on days 1, 5, and 9, i.e., intratumoral injection PBS, GOx, cGAMP, GPPM, gppm@gox, or gppm@gox+cgamp ([ cGAMP ] =5 μg/dose), respectively, until the tumor volume was about 50mm 3 (after 8 days). Meanwhile, in order to evaluate the in vivo anti-tumor immune activation of the treatment group, 5×10 5 CT26 cells were planted subcutaneously in the left leg of the mice at the time of the first treatment to construct a distal tumor, and the growth of the distal tumor was monitored for 21 consecutive days as well. Wherein the tumor volume measurement formula is as follows:
Tumor volume (V) =a×b 2/2 (1)
* A and b represent the maximum and minimum tumor diameters, respectively.
As shown in fig. 17, the mice of each group had a slow increase in body weight over time, and there was no significant difference between each group, indicating that there was no significant systemic toxicity after the material injection of each experimental group. In the case of primary tumor growth, as shown in fig. 18, the primary tumors of mice in the PBS group, the GOx group and the cGAMP group all grow rapidly, the tumor growth of mice in the GPPM experimental group is inhibited to a certain extent, the tumor growth of the mice in the GPPM@GOx and the GPPM@GOx+cgamp experimental group is obviously inhibited, wherein the tumor inhibition effect of the GPPM@GOx+cgamp experimental group is the best, the tumor of the mice in the experimental group is almost completely resolved, and the tumor inhibition rate is as high as 99.3%. In the case of the growth of the distal tumor, as shown in fig. 19, the growth of the distal tumor in the mice in the PBS group is also rapid, the growth rate of the distal tumor in the GOx group and the cGAMP group is slower than that in the PBS group, the GPPM group also has a better effect of inhibiting the distal tumor due to the stimulation of the innate immunity by Mn 2+, the gppm@gox group combines Mn 2+ with GOx, the effect of inhibiting the distal tumor is better, and simultaneously, the growth of the distal tumor in the mice is completely inhibited by implementing the chemical kinetics/starvation/immunity combined treatment in the gppm@gox+cgamp experimental group. More intuitively, as shown in fig. 20a-b, from a representative dig plot of the mouse tumor the last day of the experiment, it can be seen that the gppm@gox+cgamp experimental group had the best tumor suppression effect, with only one mouse still remaining a small primary tumor, while no mouse developed a distant tumor.
The effect of different sets of materials to elicit an immune response in vivo was further evaluated. After the experiment is finished, the spleen of the mouse is shaved, T cells in the spleen of the mouse are separated through a nylon column method, CD4 antibody carrying FITC fluorescence and CD8 antibody carrying PE fluorescence are adopted to mark the T cells, a flow cytometer is used for detection, differentiation of the CD4 and CD8+ T cells is analyzed, as shown in figure 21, the highest differentiation degree of CD8+ killer T cells in the spleen of the mouse of the GPPM@GOx+cGAMP experiment group can be found, and the highest anti-tumor immune response degree is indicated. Meanwhile, by using FOXP3 antibody carrying APC fluorescence, CD25 antibody carrying FITC fluorescence and CD4 antibody carrying PE fluorescence to mark T cells and analyzing the expression condition of immune suppression type Tregs (CD4+CD25+FOXP3+) through flow analysis, as shown in figure 22, the mice in the GPPM@GOx+cGAMP experimental group have the least population proportion of the Tregs cells, thus being more beneficial to the immune killing of organisms on tumors, and further proving that the mice in the GPPM@GOx+cGAMP experimental group have the best anti-tumor immune response effect.

Claims (9)

1. A preparation method of a dendrimer composite material comprises the following steps:
(1) Mixing the water solution of Mal-mPEG with the water solution of fifth-generation polyamide-amine PAMAM dendrimer G5, stirring in a water bath, dialyzing, purifying, and freeze-drying to obtain fifth-generation polyamide-amine PAMAM dendrimer G5-mPEG modified with methoxy polyethylene glycol mPEG;
(2) Mixing the G5-mPEG solution and the 4-bromomethyl phenylboronic acid solution, stirring in a water bath for reaction, dialyzing, purifying, freeze-drying to obtain a fifth generation polyamide-amine PAMAM dendrimer G5-mPEG-PBA modified with phenylboronic acid molecules;
(3) Adding KMnO 4 aqueous solution into the G5-mPEG-PBA aqueous solution, stirring for reaction after the dripping is finished, dialyzing, purifying and freeze-drying to obtain the dendrimer composite material G5-mPEG-PBA@MnO 2.
2. The preparation method of claim 1, wherein the molar ratio of the fifth generation polyamide-amine PAMAM dendrimer G5 to Mal-mPEG in step (1) is 1:12 to 1:15; the water bath stirring is carried out for 24-36h under the condition of 28-32 ℃.
3. The preparation method of the aqueous solution is characterized in that the solvent of the solution in the step (2) is dimethyl sulfoxide (DMSO), the molar ratio of the G5-mPEG to the 4-bromomethylbenzoic acid is 1:50-1:55, and the water bath stirring reaction is carried out for 24-36h at 68-72 ℃.
4. The preparation method of the water-based paint according to claim 1, wherein the adding of the KMnO 4 aqueous solution in the step (3) is specifically that the KMnO 4 aqueous solution is added at a constant speed through a micro-injection pump, and the flow rate of the micro-injection pump is 0.8-1.0 mL/min.
5. The method according to claim 1, wherein the step (3) comprises reacting G5-mPEG-PBA with
The molar ratio of KMnO 4 is 1:30-55, the concentration of KMnO 4 solution is 0.25-0.30 mg/mL, and the stirring reaction is carried out at room temperature for 1-1.5h.
6. The method according to claim 1, wherein the dialysis in steps (1) to (3) is carried out in ultrapure water for 1 to 3 days using a cellulose dialysis membrane having a molecular weight cut-off of 10000.
7. The dendrimer composite material prepared by the method of claim 1, wherein the composite material is a fifth generation polyamide-amine PAMAM dendrimer G5 surface modified methoxy polyethylene glycol mPEG and phenylboronic acid PBA, and manganese dioxide nano particles are wrapped inside.
8. A drug-loaded dendrimer composite material is characterized in that the dendrimer composite material prepared by the method of claim 1 is a carrier composite glucose oxidase GOx and an interferon gene stimulating factor STING agonist cGAMP.
9. Use of the drug-loaded dendrimer composite according to claim 8 for preparing a chemical kinetics/hunger/immunity combined therapeutic drug for tumor.
CN202210816497.9A 2022-07-12 2022-07-12 Dendrimer-coated manganese dioxide nanoparticle as well as preparation and application thereof Active CN115317618B (en)

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