Preparation method of liquid metal nitric oxide nanoreactor and application of liquid metal nitric oxide nanoreactor in preparation of cancer treatment drugs

Abstract

The invention discloses a preparation method of a liquid metal nitric oxide nano-reactor and application of the liquid metal nitric oxide nano-reactor in preparation of cancer treatment drugs. The liquid metal nitric oxide nano-reactor provides a cascaded self-supply system of ROS (reactive oxygen species) and NO (nitric oxide). ROS is generated by LMNDs, ROS is reserved to the maximum extent through a strategy of consuming GSH through a disulfide bond, and then guanidyl is triggered to release NO. Compared with treatment by independently using ROS and NO, the LMND-HSG nano-drug has the advantage that the treatment effect is obviously enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug research and development, and specifically relates to a method for preparing a liquid metal nitric oxide nanoreactor and an application thereof in preparing cancer therapeutic drugs. Background Art

[0002] Nitric oxide (NO) gas therapy is a promising cancer treatment method due to its mild toxicity, short treatment time, lack of drug resistance, multiple mechanisms of action (such as nitrite stress, mitochondrial / DNA damage, apoptosis, etc.), and synergistic effects with other treatment modalities such as chemotherapy, radiotherapy, photodynamic therapy, and immunotherapy. However, the direct use of NO gas or NO small molecule donors is hindered by the lipophilicity, non-selective diffusion, instability, short half-life (2-5 seconds), and susceptibility to bioactive substances such as reactive oxygen species (ROS), glutathione (GSH), and hemoglobin.

[0003] Gallium (Ga)-based liquid metal nanodroplets (LMNDs), as an emerging class of metal-based nanomaterials, have been widely studied due to their good biocompatibility and low cytotoxicity. However, LMND-based gas therapy has not been studied yet. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides a method for preparing a liquid metal nitric oxide nanoreactor, which comprises the following steps:

[0006] Step 1: preparing HPAP: dissolving 1-(2-aminoethyl)piperazine, N,N′-methylenebisacrylamide and N,N′-bis(acryloyl)cystamine in a solvent, reacting at 50-55° C. for 100-120 hours, then terminating the reaction and purifying, and drying the product to obtain HPAP;

[0007] Step 2: Preparation of HSG: Dissolve HPAP in a solvent, add 1-hydrogen-pyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine dropwise, stir at room temperature for 48-72 hours, dialyze and dry to obtain HSG;

[0008] Step 3: The gallium-indium eutectic and HSG solution were mixed, ultrasonicated in an ice bath, and centrifuged to obtain LMND@HSG.

[0009] As a preferred embodiment of the method for preparing the liquid metal nitric oxide nanoreactor described in the present invention: in step 1, the molar ratio of 1-(2-aminoethyl)piperazine, N,N′-methylenebisacrylamide and N,N′-bis(acryloyl)cystamine is (2-3):(3-4):1.

[0010] As a preferred solution of the method for preparing the liquid metal nitric oxide nanoreactor described in the present invention: in step 1, the solvent is a methanol / water mixed solvent, and the volume ratio of methanol to water is 7:3.

[0011] As a preferred embodiment of the method for preparing the liquid metal nitric oxide nanoreactor of the present invention: in step 1, the termination reaction is to add 1-(2-aminoethyl)piperazine and maintain at 37° C. for 8 to 10 hours.

[0012] As a preferred embodiment of the method for preparing the liquid metal nitric oxide nanoreactor of the present invention: in step 1, the purification includes purification by precipitation with acetone.

[0013] As a preferred embodiment of the method for preparing the liquid metal nitric oxide nanoreactor described in the present invention: in step 2, the solvent includes methanol, and the mass ratio of HPAP, 1-hydrogen-pyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine is 2-3:1:0.5-1.

[0014] As a preferred embodiment of the method for preparing the liquid metal nitric oxide nanoreactor of the present invention: in step 2, the molar ratio of 1-hydrogen-pyrazole-1-carboxamidine hydrochloride to N,N-diisopropylethylamine is 1:1.

[0015] As a preferred solution of the method for preparing the liquid metal nitric oxide nanoreactor of the present invention: in step 3, the concentration of the HSG solution is 0.41-0.42 mg / mL.

[0016] As a preferred solution of the method for preparing the liquid metal nitric oxide nanoreactor described in the present invention: in step 3, the volume concentration of the gallium-indium eutectic in the HSG solution is 0.6-0.7%.

[0017] Beneficial effects of the present invention: The present invention adopts reducible guanidine-acylated hyperbranched polyaminoamine (HSG) as a stable ligand of LNMDs through multivalent amine-gallium binding interaction. Among them, the disulfide bonds and guanidine groups in HSG consume excess glutathione (GSH) in the tumor microenvironment (TME) and act as nitric oxide (NO) donors, respectively. It is worth noting that the constructed LMND NO nanoreactor provides a cascade self-supply system for ROS and NO. ROS are generated by LMNDs, and the strategy of consuming GSH through disulfide bonds maximizes the retention of ROS, which then triggers the release of NO by guanidine groups. The therapeutic effect of LMND@HSG nanomedicine is significantly enhanced compared with the treatment with ROS and NO alone. This is the first LMND-based NO gas therapy nanoreactor. The inherent anti-tumor and anti-inflammatory activities of LMND provide a platform for combined / synergistic therapy and promote the understanding of the mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0019] Figure 1 Schematic diagram of the mechanism of LMND@HSG nanomedicine in treating cancer.

[0020] Figure 2 The synthetic route and characterization diagram of HSG ligand.

[0021] Figure 3 Synthesis and characterization diagram of LMND@HSG.

[0022] Figure 4 In vitro performance of LMND@HSG.

[0023] Figure 5 In vitro therapeutic performance of LMND@HSG.

[0024] Figure 6 The in vivo therapeutic effect of LMND@HSG.

[0025] Figure 7 1 is the H NMR spectra of HPAA and HPAP with different disulfide bond contents.

[0026] Figure 8 XPS spectrum of LMND@HSG and C1s XPS spectrum of LMND@HSG.

[0027] Fig. 9 It is the hemolysis rate of red blood cells under different concentrations of LMND@HSG.

[0028] Fig.10The CI value of the synergistic inhibitory effect of LMND@HSG on 4T1 cells was prepared by combining LMND and HSG. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0030] Experimental Materials:

[0031] Materials: Gallium-indium eutectic (EGaln, Alfa Aesar, 99.99%), cystamine hydrochloride (Sigma-aldrich, 98%), acryloyl chloride (TCI, 97%), 1-(2-aminoethyl)piperazine (AEPZ, TCI, ≥98%), N,N′-methylenebisacrylamide (MBA, Alfa, >99%), 1-hydrogen-pyrazole-1-carboxamidine hydrochloride (TCI, 98%), N,N-diisopropylethylamine (DIPEA, Alfa, 99%). Unless otherwise specified, all assay kits were purchased from Bio-Tech Biotech Co., Ltd. (Shanghai, China). 4T1 (mouse breast cancer cells) and PC3 (human prostate cancer cells) were obtained from the cell bank of Shanghai Institute of Cell Biology, China. All animal experiments were approved by the Animal Ethics Committee of Jiangsu Institute of Nuclear Medicine (No. JSINM-2023-032) and followed the corresponding guidelines.

[0032] Experimental methods:

[0033] Characterization methods: Nuclear magnetic resonance (NMR) was characterized using a Bruker Avance II 400 spectrometer. The average molecular weight of the polymer was measured on a Waters Breeze2 gel permeation chromatography (GPC) system. DMF was used as the eluent, flow rate: 1 ml / min, temperature: 35 °C. Polystyrene was used as the calibration material. The hydration kinetic size and surface potential of the nanomaterials were tested on a Marlvern Zetasizer Nano ZS90. X-ray diffraction (XRD) measurements were performed on a Bruker D8 Advance XRD diffractometer. X-ray photoelectron spectra were recorded on a Thermo ESCALAB 250XI XPS microprobe. The morphology of LMND@HSG was detected on a Tecnai G2 20 transmission electron microscope (TEM). Fluorescence probe experiments were measured on a HORIBA FluoroMax Plus fluorescence spectrometer. Cell fluorescence experiments were performed under an OLYMPUS TH4-200 inverted fluorescence microscope.

[0034] ROS in vitro detection: In deionized water, DCFH-DA probe (10 μM) and LMND@HSG were mixed in different concentrations of H2O2 (0.1 mM, 5 mM, 10 mM) at 37 °C in the dark for 1 h. After centrifugation (6000 rpm, 5 min), the samples were detected on a fluorescence spectrometer with an excitation wavelength of 488 nm (Ex488) and an emission wavelength of 525 nm (Em525).

[0035] Cells: 4T1 cells were seeded in 24-well plates (2×10 4 The cells were then cultured with 10 μg / ml HSG, 40 μg / ml LMND, and 40 μg / ml LMND@HSG, respectively. After another 4 h of incubation, the cells were rinsed three times with PBS and reacted with DCFH-DA (10 μM) for 40 min. Finally, the excess DCFH-DA was removed, the cells were stained with Hoechst 33342 for 3 min, and the cell fluorescence was recorded under a fluorescence microscope.

[0036] NO in vitro detection: In deionized water, HSG (0.5 mg / ml) and LMND@HSG (2 mg / ml) were reacted with 10 mM H2O2 or without 10 mM H2O2 at 37 °C for 24 h under the DAF FM-DA probe (5 μM). The collected supernatants were analyzed by fluorescence spectrometer. x 495 and E m 515 was measured.

[0037] Cells: 4T1 cells (2 × 10 per well) were cultured in 24-well plates. 4 The cells were then cultured with 10μg / ml HSG, 40μg / ml LMND, and 40μg / ml LMND@HSG, respectively. After incubation for 4h, the cells were rinsed with PBS and reacted with 5μM DAF-FM DA for 24h. Finally, they were washed with PBS three times to remove free DAF-FM DA, and stained with Hoechst 33342 for 3min, and the cell fluorescence was recorded under a fluorescence microscope.

[0038] In vitro reaction of HSG with glutathione: 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) is an Ellman reagent for determining the thiol content in the target molecule to analyze the consumption of GSH. Briefly, glutathione (2 mM) was mixed with an equal amount of sample solution (HSG: 2 mg / ml) at room temperature for 12 hours. 6.6 μL of DTNB aqueous solution was diluted with 150 μL Tris-HCl and mixed with the above reaction mixture for 15 minutes, and finally the reaction components were identified by high performance liquid chromatography (HPLC).

[0039] Immunofluorescence of DNA double-strand breaks: γ-H2AX immunofluorescence staining was used to study DNA double-strand breaks. Briefly, 4T1 cells were seeded in 24-well plates (2 × 10 4 / well), and then added 10μg / ml HSG, 40μg / ml LMND and 40μg / ml LMND@HSG respectively. After incubation for 24h, the treated cells were rinsed with PBS and incubated with 4% paraformaldehyde. The fixed cells were treated with 0.25% Triton X-100 and incubated with immunofluorescence blocking buffer for 20min. The γ-H2AX rabbit monoclonal antibody (C2036S, Biyuntian Biotechnology Co., Ltd.) was incubated with the cells at 4°C overnight. The secondary antibody was incubated with the cells at room temperature for 1h, and the cell nucleus was stained with Hoechst 33342. Finally, the cell fluorescence was observed under a fluorescence microscope.

[0040] Intracellular mitochondrial damage detection: 4T1 cells were seeded in 24-well plates (2×10 4 The cells were then cultured with 10 μg / ml HSG, 40 μg / ml LMND, and 40 μg / ml LMND@HSG, respectively. After 24 h of incubation, the cells were rinsed three times with PBS and cultured with Mito-Tracker probe (Biyuntian Biotechnology Co., Ltd.) for 30 min. Flow cytometry and fluorescence microscopy were used to observe mitochondrial damage in the cells.

[0041] Cytotoxicity of LMND@HSG on 4T1 cells: 4T1 cells were plated in 96-well plates (6 × 10 3 After incubating the cells with different concentrations (0, 2, 5, 10, 20, 40, 80, 160, 320 μg / ml) of LMND@HSG for 24 h, 10 μl of CCK-8 solution was added and incubated for another 30 min. Finally, the absorbance at 450 nm was recorded on a microplate reader.

[0042] Effect of LMND@HSG on apoptosis of 4T1 cells: Live / dead cell viability assay: 4T1 cells were seeded in 24-well plates (2×10 4 The cells were incubated with 40 μg / ml HSG, 160 μg / ml LMND and 40 μg / ml LMND@HSG for 24 h, and then incubated with Calcein-AM and propidium iodide (PI) for 30 min. The stained cells were observed under a fluorescence microscope.

[0043] Annexin V-FITC / PI detection: 4T1 cells were seeded in 6-well plates (1×10 524 hours later, 40μg / ml HSG, 160μg / ml LMND, and 40μg / ml LMND@HSG were added to the cells for co-incubation. After 24 hours, the cells were collected by trypsin. Finally, the collected cells were incubated with Annexin V-FITC / PI in a centrifuge tube for 30 minutes and detected by flow cytometry. The data were processed by FlowJo V10 software.

[0044] Animal model: All animal experimental protocols used in this study were approved by the Animal Care and Ethics Committee of Jiangsu Institute of Atomic Medicine. Female BALB / c mice (5-6 weeks, 18-22 g body weight) were purchased from Cavens Laboratory Animal Co., Ltd., Changzhou, China. Luciferase-labeled 4T1 (4T1-Iuc) cells (1×10 6 The 4T1 mouse model was established by subcutaneously injecting 4T1 cells into the right side of female BALB / c mice. Tumor volume calculation formula (mm 3 ) is 0.5×length×width 2 When the tumor volume approaches 100mm 3 Around , mice were randomly divided into predetermined groups for further experiments.

[0045] In vivo efficacy: To evaluate the therapeutic effect of LMND@HSG in vivo, 2 mg / kg HSG, 2 mg / kg LMND, and 2 mg / kg LMND@HSG and saline were injected into 4T1 tumor-bearing mice every other day. After 15 days of treatment or when the tumor volume was close to 1000 mm 3 At 4 pm, the mice were killed, and the solid tumors and major organs (heart, liver, spleen, lung, and kidney) were isolated for further analysis and characterization.

[0046] Statistical analysis: SPSS 24.0 statistical analysis software was used for statistical processing. The results are expressed as mean ± standard deviation (sd) (n ≥ 3). For comparisons between two groups and multiple groups, two-tailed Student's t test and one-way analysis of variance (ANOVA) were used, respectively. P < 0.05 was considered statistically significant, and ns was considered not statistically significant.

[0047] Embodiment 1:

[0048] (1) Synthesis of N, N'-bis(acryloyl)cystamine (CBA):

[0049] The synthesis method of CBA is as follows: Cystamine hydrochloride (6.80g, 30.0mmol) and sodium hydroxide (5.20g, 130.0mmol) are dissolved in 150ml of deionized water, and then acryloyl chloride (6.40g, 70.0mmol, dissolved in 6ml of chloroform) is added dropwise. The reaction mixture is stirred at 0°C for 6h. The raw material is filtered and rinsed three times with deionized water. The mixed solution is then extracted with dichloromethane (3×100ml). The organic phase is then dried with anhydrous sodium sulfate, and the dichloromethane is then removed in vacuo at room temperature. Finally, CBA is purified by recrystallization with ethyl acetate, and the yield is 67.4%.

[0050] (2) Synthesis of hyperbranched polyamidoamine (HPAA) and bioreducible HPAP (reduced polyamidoamine):

[0051] HPAA and HPAP containing disulfide groups were prepared by Michael addition reaction:

[0052] Preparation of hyperbranched polyamine (HPAA): AEPZ (258.3 mg, 2.00 mmol) and MBA (616.9 mg, 4.00 mmol) (AEPZ / MBA = 1:2) were added to a 20 ml vial and dissolved in a methanol / water mixed solvent (8.0 ml, v / v = 7 / 3). The mixture was placed at 55°C for 120 h, and then the acrylamide group of HPAA was terminated with AEPZ (361.7 mg, 2.80 mmol) at 37°C for 8 h. The raw material was purified by precipitation in cold acetone (0°C) and vacuum dried at room temperature for 2 h to obtain HPAA.

[0053] Preparation of HPAP: AEPZ (258.4 mg, 2.00 mmol), MBA (462.7 mg, 3.00 mmol) and CBA (260.4 mg, 1.00 mmol) (AEPZ / MBA / CBA = 2:3:1) were added to a 20 ml vial and dissolved in a methanol / water mixed solvent (8.0 ml, v / v = 7 / 3). The mixture was placed at 55°C for 120 h, and then the acrylamide group of HPAP was terminated with AEPZ (361.7 mg, 2.80 mmol) at 37°C for 8 h. The raw material was purified by precipitation in cold acetone (0°C) and dried under vacuum at room temperature for 2 h.

[0054] (3) Preparation of HSG (polyamidoamine guanidine) by guanylation reaction of hyperbranched polyamidoamine:

[0055] HPAP (1.0 g) was dissolved in 15 ml of methanol, and then 1-hydrogen-pyrazole-1-carboxamidine hydrochloride (410.4 mg, 2.80 mmol) and DIPEA (361.9 mg, 2.80 mmol) were added dropwise. The reaction mixture was stirred at room temperature for 48 h, dialyzed in cold deionized water (4°C) for 72 h, and finally freeze-dried and collected to obtain HSG.

[0056] (4) Preparation of LMND@HSG:

[0057] The gallium-indium eutectic EGaln (80 μL) and the aqueous solution of HSG obtained in step (3) (5 mg, 12 ml) were added to a centrifuge tube, ultrasonicated in an ice bath for 12 min, centrifuged at 1200 rpm for 5 min, the precipitate was discarded, and the suspension was collected to obtain LMND@HSG, and the concentration was determined by freeze-drying method.

[0058] Experimental results:

[0059] Synthesis of NO donor HSG ligand:

[0060] like Figure 2 As shown in Figure A, different molar ratios of bisacrylamide (MBA and CBA, n / n: 1 / 0, 5 / 1, 4 / 1, 3 / 1) and APEZ (half the molar amount of bisacrylamide) were subjected to Michael addition reaction in a mixed solvent of water and methanol (v / v: 7 / 3). Different ratios of CBA and MBA can adjust the content of disulfide bonds, thereby changing the hydrophilicity, bioreducibility and solubility of the polymer. In addition, APEZ was used to terminate the acrylamide end groups to obtain HPAA and HPAP. Since different polymerization conditions, such as molar feed ratio, solvent, temperature, etc., can change the topological structure of HPAA, the reaction conditions are strictly controlled. The structures of HPAA and HPAP were verified by nuclear magnetic resonance hydrogen spectrum ( Figure 7 ). The typical proton signal of the double bond of acrylamide disappeared, indicating that the AEPZ end-capping was complete. With the increase of CBA addition ratio (HPAP31>HPAP41>HPAP51>HPAA10), the corresponding HPAP methylene proton signal at 2.78 ppm (marked by blue band) in the H NMR spectrum increased, with the MBA methylene proton at 4.35 ppm (marked by red band) as a reference. In order to further connect the guanidine group at the end of HPAA / HPAP, HPAA / HPAP was guanylated with 1H-pyrazole-1-monohydrochloride formamidine, and the resulting product HSG had a typical guanidine proton signal of 7.83-8.35 ppm (marked by cyan band) in the H NMR spectrum ( Figure 2 B). Gel permeation chromatography (GPC) curves show that the molecular weight of HSG increases after guanidation ( Figure 2 C). In addition, the infrared spectrum showed that 1218 and 1708 cm-1 The CO stretching and C=O vibration bands at 1530 to 1522 cm -1 The blue shift indicates that -NH2 + Bending vibration peak formation ( Figure 2 D).

[0061] Synthesis and characterization of LMND@HSG:

[0062] Ligands play an important role in stabilizing LMNDs. HPAA can stabilize LMNDs through surface absorption of tertiary amines. We introduced disulfide bonds and guanidine groups into the HPAA structure, namely HSG. The HSG ligand has the effect of stabilizing LMND and releasing NO under ROS stimulation. At the same time, the presence of disulfide bonds can consume glutathione, thereby increasing the retention of reactive oxygen species. The synthesis method of LMND@HSG is as follows Figure 3 As shown in A. TEM images show that LMND@HSG has a spherical morphology and is covered with a polymer layer ( Figure 3 B, Figure 3 C), and the overall size is 186.0±16.4nm. DLS measurements show that the hydration dynamic size of LMND@HSG is 212.7±2.6nm, the zeta potential is +34.20mV, and the PDI is 0.11( Figure 3 D). During the 7-day monitoring, the hydration dynamic size and zeta potential did not change significantly, indicating that LMND@HSG has good stability ( Figure 3 E).

[0063] In order to verify the composition of LMND@HSG, FTIR and XRD tests were performed. Figure 3 F), the similar spectra of HSG and LMND@HSG indicate the presence of HSG in the nanocomposite. The XRD spectrum of LMND@HSG consists of separate PDF cards of gallium oxyhydroxide (GaO(OH)) and indium (In), confirming that the composition of LMND@HSG is consistent with previous research results ( Figure 3 G). The interaction between LMND and HSG was further investigated by XPS analysis. In addition to the main elements gallium, indium, carbon, oxygen and nitrogen ( Figure 8 In addition to the Ga 3D high-resolution XPS spectrum, two signals with binding energies (BE) of 17.3 eV and 19.5 eV are attributed to Ga (0 valence) and Ga (3 valence), respectively, indicating that a Ga oxide layer is formed on the LMNDs. Figure 3 H). In the N1s spectrum, the three peaks BEs at 399.1, 396.7 and 393.6 eV belong to the characteristic peaks of NH, N-Ga(III) and Ga LMM, respectively ( Figure 3l). Under C1s conditions, the three deconvoluted peaks at BEs of 284.6 eV (CC), 285.6 eV (Cn), and 287.4 eV (C=O) perfectly match the carbon bond types in HSG ( Figure 8 B). Taken together, these results verify that HSG is bound to the surface of LMNDs via N-Ga, resulting in the synthesis of well-defined LMND@HSG nanocomposites.

[0064] In vitro ROS and NO release of LMND@HSG:

[0065] like Figure 4 As shown in A, LMND@HSG containing guanidine groups can trigger the generation of ROS under acidic conditions, while the disulfide bonds within the HSG skeleton can consume GSH in the TME, thereby reducing the consumption of ROS. Therefore, this cascade system can promote the supply of ROS and the production of NO, providing conditions for the combination of ROS-related chemotherapy and gas therapy. We used DCFH-DA and DAF-MFDA probes to detect the ability of LMND@HSG to produce ROS and NO, respectively. In the ROS test, H2O2 alone (0.1, 5, 10mM) did not produce fluorescence, but co-incubated with LMND@HSG produced fluorescence, indicating that ROS was generated ( Figure 4 B). The higher the H2O2 concentration, the stronger the fluorescence intensity (FI). In contrast, incubation of LMND@HSG alone without H2O2 produced only weak fluorescence. These results indicate that ROS mainly originate from LMND, and the low pH of H2O2 induces LMND degradation and accelerates the generation of ROS.

[0066] GSH is an important factor in regulating redox balance and can consume ROS. Therefore, a disulfide bond was introduced into the HSG structure to consume endogenous GSH in the TME. The glutathione and glutathione disulfide (GSSG) kit was used to measure the glutathione consumption capacity of HSG. GSH can react with the chromogenic substrate DTNB to generate yellow 2-nitro-5-thiobenzoic acid (TNB) and GSSG, and the composition of the reaction mixture was monitored by high performance liquid chromatography. Figure 4 As shown in C and D, HSG10 can hardly consume GSH (0.2%). With the increase of disulfide bond content, GSH consumption increased, among which HSG31 had the highest consumption of 11.5%. When 1 mM GSH was added, the ROS content of LMND@HSG group and LMND@HSG10 group decreased to 35.5% and 11.4%, respectively ( Figure 4E). Due to the presence of disulfide bonds, LMND@HSG retains more reactive oxygen species than LMND@HSG10. To demonstrate whether the retention of ROS can trigger the generation of NO in LMND@HSG, we reacted 5 μM DAF-MF DA with HSG, HSG+10 mM H2O2, LMND@HSG, and LMND@HSG+10 mM H2O2 aqueous solutions. Figure 4 As shown in F, the LMND@HSG+10mM H2O2 group m The FI at 525 was the highest, indicating that HSG could generate NO more effectively under the stimulation of LMND and H2O2.

[0067] Next, the production of ROS and NO at the cellular level was investigated by confocal laser scanning microscopy (CLSM). Figure 4 As shown in Figure G, the LMND@HSG incubation group had the strongest cell fluorescence in the DCFH-DA channel, while in the LMND incubation group, the expected fluorescence could be observed due to its energy storage properties. In contrast, no green fluorescence was detected in the HSG group and the control group. Similarly, NO cell fluorescence studies showed that in the DAF-MF DA channel, only the LMND@HSG incubation group had green fluorescence production, indicating that the insufficient ROS level in the TME can be increased by supplementing LMND, thereby triggering NO production ( Figure 4 H). Flow cytometry experiments also confirmed this trend, with the LMND@HSG group having the highest percentage of positive cells (DAF-MF DA staining) ( Figure 4 I).

[0068] In vitro cytotoxicity of LMND@HSG:

[0069] The cytotoxicity of LMNG@HSG was evaluated by the methyl tetrazolium (MTT) method. When LMNG and HSG were used alone, they still had weak cytotoxicity to 4T1 and PC-3 cells at a concentration of up to 160 μg / ml (cell viability > 73%) ( Figure 5 A), indicating that they have good compatibility. In contrast, LMND@HSG showed obvious cytotoxicity, and the half inhibitory concentration (IC50) value of LMND@HSG on 4T1 cells was 29.76μg / ml. The synergistic inhibitory effect of LMND@HSG prepared by combining LMND and HSG on 4T1 cells was shown in the CI value statistics. Fig.10 .from Fig.10It can be seen that the Cl value is less than 1, indicating that the combination of LMND and HSG has a significant synergistic effect. Calcein AM / PI staining and Annexin V-FITC apoptosis kit flow cytometry were used to detect the in vitro cytotoxicity of LMND@HSG. At a drug concentration of 40μg / ml, Calcein AM / PI staining results showed that the LMND@HSG incubation group showed obvious red fluorescence (>89%), indicating that it has high cytotoxicity; and under LMND and HSG treatment, most 4T1 cells (96.3%, 97.8%) still survived ( Figure 5 B). Annexin V-FITC cell apoptosis assay showed that the apoptosis rate of LMND@HSG cells (52.9%) was significantly higher than that of other groups (LMND: 12.4%; HSG: 24.5%; control group: 9.1%) ( Figure 5 C), these results are consistent with those of the MTT assay.

[0070] When the concentration is greater than 1 μM, NO can damage mitochondria and DNA through nitration structure, inhibiting tumor growth and metastasis. Mito-Tracker Red CMXRos can stain normal mitochondria with strong red fluorescence, and FI is negatively correlated with mitochondrial membrane potential, so the occurrence of apoptosis is usually accompanied by the weakening or quenching of red fluorescence. Figure 5 As shown in D, the reduction of red fluorescence indicates that mitochondrial damage basically occurred in the LMND@HSG group, and it was not obvious in other groups. The DNA damage detection kit uses γ-H2AX immunofluorescence to detect the degree of DNA damage. The green fluorescence of the LMND@HSG group is the strongest, indicating that DNA damage is obvious ( Figure 5 E). The mean fluorescence intensity (MFI) of Mito-Tracker and γ-H2AX in the LMND@HSG group was significantly different from that in the other groups ( Figure 5 F). These results suggest that HSG@LMND nanomedicine can release ROS and NO in tumor cells, leading to DNA damage and mitochondrial dysfunction.

[0071] In vivo therapeutic effects of LMND@HSG:

[0072] The in vivo anticancer therapeutic effect of LMND@HSG was evaluated in a 4T1 tumor-bearing BALB / c nude mouse model. No obvious red blood cell hemolysis was detected after interaction with LMND@HSG ( Fig. 9 ). Since systemic administration can cause severe toxicity and promote new metastasis of breast cancer cells, this study used intratumoral injection for further treatment. The mice were divided into 5 groups: LMND group, HSG group, LMND@HPAA group, LMND@HSG group and saline control group. These mice were topically administered with the same concentration of 2 mg / kg ( Figure 6A) Tumor growth was assessed by caliper measurement. Figure 6 As shown in C, the tumor growth in the LMND@HSG-treated group was significantly inhibited, while single treatment of LMND, HSG, and LMND@HPAA groups as controls had almost no effect. The IVIS imaging system on days 1, 4, 7, 9, and 11 after treatment also confirmed that LMND@HSG administration had the best antitumor effect ( Figure 6 B). During the administration period, there was no significant change in the body weight of mice in each group, indicating that the components of LMND@HSG have good biocompatibility ( Figure 6 D).

[0073] On the 12th day after treatment, when the tumor volume exceeded 1000mm 3 At 14:00, the mice were euthanized, and the tumor tissues and major organs (heart, liver, spleen, lung, and kidney) of each group were collected for further characterization. H&E staining results showed that no damage or lesions were found in the major organs ( Figure 6 E) In addition, TUNEL staining was performed on the excised tumors to assess the extent of cell necrosis and apoptosis. Figure 6 E and F show that LMND@HSG treatment resulted in the most severe local tumor necrosis and the most obvious DNA damage by γ-H2AX staining. In contrast, LMND@HPAA only caused partial cell apoptosis without obvious DNA damage, which may be due to the involvement of only ROS but no NO in the treatment. In addition, immunohistochemistry results of Ki-67 and vascular endothelial growth factor (VEGF) showed that tumor proliferation and angiogenesis were significantly inhibited after LMND@HSG treatment. These data indicate that the combined release of ROS and NO by LMND@HSG nanomedicine can significantly improve the therapeutic effect of tumors.

[0074] In summary, we developed a TME-responsive LMND-based gas nanoreactor for NO-enhanced tumor therapy. Disulfide bonds initiated GSH depletion and decomposition of HSG ligands in the TME, subsequently activating the degradation of LMND and leading to the cascade generation of ROS and NO. Along with the inherent anticancer activity of LMND, the LMND@HSG nanoprodrug constructed using both ROS and NO enhanced the therapeutic effect. In addition, the inherent anti-inflammatory properties of LMND distinguished this ROS-based nanomedicine from previous studies, and its simple composition and multifunctionality made this LMND-based nanomedicine promising for synergistic therapy.

[0075] Figure 1Schematic diagram of the mechanism of LMND@HSG nanomedicine for cancer treatment. LMND@HSG can generate ROS in TME, consume endogenous GSH, and promote ROS retention. The enhanced accessibility of ROS in TME will promote the release of NO for combined cancer therapy, while causing DNA double-strand breaks and mitochondrial dysfunction, ultimately leading to tumor cell apoptosis.

[0076] Figure 2 The synthetic route and characterization diagram of HSG ligand. (A) Structure of HSG with different disulfide bond contents. (B) H NMR spectrum of HSG with different disulfide bond contents. (C) GPC curves of HPAP and HSG with different disulfide bond contents. (D) FTIR spectrum of HPAP and HSG with different disulfide bond contents.

[0077] Figure 3 Synthesis and characterization of LMND@HSG. (A) Schematic diagram of the synthesis of LMND@HSG. (B, C) TEM images of LMND@HSG, scale bar 1μm (B), 50nm (C). (D) Particle size, PDI and surface potential of LMND@HSG (40μg / ml in water) were determined by DLS method. All data are expressed as mean±sd, n=4. (E) Particle size and surface potential stability of LMND@HSG in water for 7 days. (F) FTIR spectra of LMND@HSG and HSG. (G) XRD spectra of LMND@HSG, In and GaOOH. (H, I) High-resolution Ga 3d and N1s XPS spectra of LMND@HSG.

[0078] Figure 4 In vitro performance of LMND@HSG. (A) Schematic diagram of LMND@HSG activating ROS and NO generation. (B) ROS generation of LMND@HSG under or without H2O2 stimulation. (C, D) Quantitative analysis of GSH consumption capacity of HSG by HPLC using DTNB as substrate. (E) ROS generation / retention of LMND@HSG in the presence of 1 mM GSH. (F) Effects of LMND and H2O2 on the NO generation / retention capacity of HSG ligand. (G) ROS generation test of 4T1 cell HSG, LMND and LMND@HSG mediated by DCFH-DA probe, scale bar, 50 μm. (H) NO generation test of 4T1 cell HSG, LMND and LMND@HSG mediated by DAF-MF DA probe, scale bar, 50 μm. (I) Flow cytometry detection of NO generation of HSG, LMND and LMND@HSG mediated by DAF-MF DA probe.

[0079] Figure 5In vitro therapeutic properties of LMND@HSG. (A) In vitro cytotoxicity of LMND, HSG and LMND@HSG on 4T1 and PC3 cells for 24 h. All data are presented as mean±sd, n=4. ＊ P<0.05, ＊＊＊ P<0.001. (B) Live / dead staining experiment of 4T1 cells after incubation with LMND, HSG and LMND@HSG for 24h. Live cells and dead cells were stained with green and red fluorescence, respectively. Scale bar, 50μm. (C) LMND@HSG-induced apoptosis of 4T1 cells for 24h was analyzed by flow cytometry using Annexin V-FITC / PI staining. (D) Mitochondrial damage in HSG, LMND and LMND@HSG-incubated 4T1 cells was detected by Mito-Tracker (200nM). Scale bar, 50μm. (E) γ-H2AX immunofluorescence was used to detect DNA damage in HSG, LMND and LMND@HSG-incubated 4T1 cells. Scale bar, 50μm. (F) Figure 5 MFI analysis of Mito-Tracker and γ-H2AX fluorescence staining in D and E. All data are presented as mean ± SD, n = 3. ＊＊＊ P < 0.001, ＊＊＊＊ P<0.0001.

[0080] Figure 6 The in vivo therapeutic effect of LMND@HSG. (A) Schematic diagram of in vivo treatment of 4T1 xenograft mouse model. (B) Dynamic |V|S imaging of 4T1-luc tumor mice after different treatments. (C, D) Average tumor growth curves and body weights of mice treated with saline, LMND (2 mg / kg), HSG (2 mg / kg), LMND@HPAA (2 mg / kg), and LMND@HSG (2 mg / kg). All data are expressed as mean±sd, n=6. ＊＊＊＊ P < 0.0001. (E) H&E staining of major organs and tumors. TUNEL staining, γ-H2AX staining, Ki67, and VEGF immunohistochemical staining of 4T1 tumor sections. Scale bar, 100 μm. (F) Statistical analysis of immunohistochemical parameters (Ki-67 and VEGF) and MFI detected by TUNEL and γ-H2AX, n = 3. All data are expressed as mean ± sd. ＊＊ P<0.01, ＊＊＊＊ P<0.0001.

[0081] Figure 7 1 is the H NMR spectra of HPAA and HPAP with different disulfide bond contents.

[0082] Figure 8 (A) XPS spectrum of LMND@HSG. (B) C1s XPS spectrum of LMND@HSG.

[0083] Fig. 9 The red blood cell hemolysis rate under different concentrations of LMND@HSG (2%, v / v). All data are expressed as mean±sd, n=3.

[0084] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a liquid metal nitric oxide nanoreactor, characterized in that: The following steps are included: Step 1: Preparation of HPAP: dissolving 1-(2-aminoethyl)piperazine, N,N′-methylenebisacrylamide and N,N′-bis(acryloyl)cystamine in a solvent, reacting at 50-55° C. for 100-120 h, then terminating the reaction and purifying, and drying the product to obtain HPAP; Step 2: Preparation of HSG: Dissolve HPAP in a solvent, add 1-hydrogen-pyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine dropwise, stir at room temperature for 48-72 hours, dialyze and dry to obtain HSG; Step 3: The gallium-indium eutectic and HSG solution were mixed, ultrasonicated in an ice bath, and centrifuged to obtain LMND@HSG.

2. The method for preparing a liquid metal nitric oxide nanoreactor according to claim 1, characterized in that: In step 1, the molar ratio of 1-(2-aminoethyl)piperazine, N,N′-methylenebisacrylamide and N,N′-bis(acryloyl)cystamine is (2-3):(3N4):

1.

3. The method for preparing a liquid metal nitric oxide nanoreactor according to claim 1 or 2, characterized in that: In step 1, the solvent is a methanol / water mixed solvent, and the volume ratio of methanol to water is 7:

3.

4. The method for preparing a liquid metal nitric oxide nanoreactor according to claim 1 or 2, characterized in that: In step 1, the termination reaction is performed by adding 1-(2-aminoethyl)piperazine and maintaining at 37° C. for 8 to 10 hours.

5. The method for preparing a liquid metal nitric oxide nanoreactor according to claim 1 or 2, characterized in that: In step 1, the purification includes purification by precipitation with acetone.

6. The method for preparing a liquid metal nitric oxide nanoreactor according to claim 1 or 2, characterized in that: In step 2, the solvent includes methanol, and the mass ratio of HPAP, 1-hydrogen-pyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine is 2-3:1:0.5-1.

7. The method for preparing a liquid metal nitric oxide nanoreactor according to claim 1 or 2, characterized in that: In step 2, the molar ratio of 1-hydrogen-pyrazole-1-carboxamidine hydrochloride to N,N-diisopropylethylamine is 1:

1.

8. The method for preparing a liquid metal nitric oxide nanoreactor according to claim 1 or 2, characterized in that: In step 3, the concentration of the HSG solution is 0.41-0.42 mg / mL.

9. The method for preparing a liquid metal nitric oxide nanoreactor according to claim 1 or 2, characterized in that: In step 3, the volume concentration of gallium-indium eutectic in the HSG solution is 0.6-0.7%.

10. Use of the liquid metal nitric oxide nanoreactor prepared by the preparation method according to claim 1 in preparing cancer therapeutic drugs.

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