Nano-particles containing biological enzyme as well as preparation method and application of nano-particles

Abstract

The invention discloses nano-particles containing biological enzyme as well as a preparation method and application of the nano-particles. The double-drug nano-particle provided by the invention is prepared by taking a nano composite material Cu / ZIF-8 obtained by modifying a ZIF-8 carrier with a copper base as a carrier, wrapping PPE in the carrier, adsorbing a small molecule drug CB-839 outside to form a nano-drug, and modifying the nano-drug with hyaluronic acid. The double-drug nanoparticle improves the properties of free PPE and CB-839, improves the stability, prolongs the tumor residence time and enhances the curative effect; and PPE and CB-839 can be released under low pH response of lysosome in cells, so that high-efficiency and low-toxicity treatment of tumors is realized. The double-drug nanoparticle can achieve a synergistic anti-tumor effect that 1 + 1 is greater than 2, and has a good application prospect in preparation of anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the field of nanotechnology, and specifically relates to nanoparticles containing biological enzymes, and a preparation method and application thereof. Background Art

[0002] Malignant tumors are one of the major diseases that seriously endanger human health and are one of the main causes of death worldwide. In recent decades, the diagnosis and treatment of tumors have experienced milestone developments, from chemotherapy to tumor targeted therapy and nanomedicine.

[0003] Reactive oxygen species (ROS) generally refers to superoxide anion free radicals (O2 ·- ), hydrogen peroxide (H2O2), singlet oxygen ( 1 ROS are highly reactive oxygen-converting substances such as iodine (O2), hypochlorous acid (HClO), and hydroxyl radicals (·OH). Appropriate concentrations of reactive oxygen species are essential for normal cell growth and survival, while elevated reactive oxygen species and impaired reactive oxygen species scavenging capacity can lead to a disorder of reactive oxygen homeostasis, further leading to amplified oxidative stress. ROS play a double-edged sword role in tumor development. A moderate increase in ROS levels is beneficial to maintaining a high proliferation rate of cancer cells, while excessive ROS can kill cancer cells by destroying cellular components, including lipids, proteins, and nucleic acids (DNA and RNA). Therefore, selectively regulating ROS levels in tumor tissues is considered to be another strategy to combat cancer.

[0004] Recent studies have shown that neutrophil elastase (ELANE) or its homologue (porcine pancreatic elastase (PPE)) released by neutrophils can kill multiple types of cancer cells (35 different cancer cell lines). Mechanistically, ELANE or PPE enters cells and proteolytically liberates the CD95 death domain (DD). CD95 DD then induces DNA damage, triggering the nuclear cytoplasmic translocation of histone H1 isoforms, which bind to CD95 DD and subsequently co-localize with mitochondria, generating a large amount of ROS in mitochondria, and finally initiating the mitochondrial apoptosis pathway.

[0005] To date, several cancer treatments related to ROS generation have been developed, including chemotherapy, radiotherapy, photodynamic therapy (PDT), sonodynamic therapy (SDT), chemodynamic therapy (CDT), etc. Compared with the above other ROS-related therapies, CDT has great potential in cancer treatment because the TME is characterized by overexpression of hydrogen peroxide (100 μM-1 mM) and mild acidity (pH ≈ 6.5), which not only facilitates the Fenton reaction but also avoids damage to normal tissues. 2+ 、Co 2+ , Mn 2+ , Cu2+ Various metal ions such as hydroxyl radicals can be used as Fenton's reagent to convert mild H2O2 into cytotoxic ·OH for CDT, ultimately inducing tumor cell apoptosis.

[0006] However, in order to balance the accumulation of excessive ROS, tumor cells produce more antioxidants (such as GSH) to maintain redox homeostasis in the tumor microenvironment (TME). This glutathione-related antioxidant defense system limits the catalytic effect of nanozymes, making it impossible to achieve the expected anti-tumor therapeutic effect. In addition, the GSH consumption efficiency of current nanocatalysts designed based on GSH scavenging is relatively low, and nanocatalysts can only play an auxiliary role in anti-tumor therapy. In order to improve the performance of GSH consumption, in addition to consuming the existing GSH in tumor cells, it is also necessary to block the endogenous GSH synthesis pathway (such as BSO, V-9302, CB-839, etc.). Therefore, it is necessary to regulate the TME by exploring strategies to promote GSH depletion to achieve more effective tumor catalytic therapy.

[0007] Therefore, finding a suitable PPE delivery carrier, improving the drugability of PPE, achieving multi-target GSH consumption, and maximizing oxidative damage may become the preferred solution to improve the clinical effect of PPE. Summary of the invention

[0008] The present invention aims at the deficiencies of the prior art and provides a nanoparticle containing a biological enzyme and a preparation method and application thereof. The nanoparticle containing the biological enzyme is a nano drug delivery system using Cu / ZIF-8 as a carrier to load a neutrophil expression enzyme and a glutathione synthesis inhibitor, and has dual glutathione depletion properties.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention claims protection for a pharmaceutical composition comprising a neutrophil-expressed enzyme and a glutathione synthesis inhibitor, wherein the neutrophil-expressed enzyme is at least one of neutrophil elastase (ELANE) and porcine pancreatic elastase (PPE); and the glutathione synthesis inhibitor is at least one of BSO, V-9302 and CB-839.

[0011] Furthermore, the mass ratio of the neutrophil-expressed enzyme to the glutathione synthesis inhibitor is 1:100 to 100:1.

[0012] In the second aspect, the present invention claims protection for a dual-drug nanoparticle, which is a nanocomposite material Cu / ZIF-8 obtained by using a copper-based modified ZIF-8 carrier as a carrier, and a nanomedicine formed by encapsulating a neutrophil-expressed enzyme inside the carrier and adsorbing a glutathione synthesis inhibitor on the outside; the neutrophil-expressed enzyme is at least one of neutrophil elastase (ELANE) and porcine pancreatic elastase (PPE); the glutathione synthesis inhibitor is at least one of BSO, V-9302 and CB-839.

[0013] As a preferred technical solution, the surface of the dual-drug nanoparticles is also modified with hyaluronic acid.

[0014] The dual-drug nanoparticles of the present invention are an anti-tumor nanomedicine that induces multiple mechanisms of tumor cell death. The dual-drug nanoparticles use Cu / ZIF-8 as a carrier, the interior of the carrier encapsulates a neutrophil-expressed enzyme (such as ELANE or PPE) that has tumor cell-specific killing properties, and the exterior adsorbs a small molecule drug of a glutathione synthesis inhibitor (such as BSO, V-9302 or CB-839) to form a nanomedicine. The synergistic anti-tumor effect of the two drugs is more significant, and the modification of hyaluronic acid can enhance its tumor targeting.

[0015] The PPE is a homologue of neutrophil elastase (ELANE) released by human neutrophils, and PPE is similar to ELANE in its ability to cleave CD95 and selectively kill cancer cells. However, because PPE is more resistant to serine protease inhibition, its catalytic activity is retained in the TME and its anticancer efficacy is significantly improved. In addition, the combination of the glutathione synthesis inhibitor CB-839 and the copper-based modification of the ZIF-8 carrier can amplify the efficacy of PPE in tumor cells and activate the immunogenic cell death of tumor cells. The nanocarriers encapsulating the two therapeutic drugs can achieve a large amount of cellular endocytosis, and can help the drugs escape from the lysosomes and release Cu in the cytoplasm. 2+ , PPE and CB-839.

[0016] Furthermore, the Zn in the nanocomposite Cu / ZIF-8 2+ With Cu 2+ The molar ratio of Zn in the ZIF-8 carrier is 2-4:1; 2+ The molar ratio of 2-methylimidazole is 1:20 to 100. Under this ratio, the co-coordination forms nanoparticles with suitable size and uniform dispersion, which can better achieve high-efficiency and low-toxicity treatment of tumors.

[0017] In a third aspect, the present invention claims a method for preparing the above-mentioned dual-drug nanoparticles, which is:

[0018] The neutrophil-expressed enzyme and zinc nitrate solution were added to the 2-methylimidazole solution, stirred for reaction, centrifuged and the supernatant was discarded, glucose solution was added for resuspending, and then ultrasonic homogenization was performed to obtain ZIF-8 nanoparticles encapsulating the neutrophil-expressed enzyme;

[0019] Adding copper chloride solution to the ZIF-8 nanoparticles for copper-based modification, stirring for reaction, centrifuging and discarding the supernatant, adding glucose solution for resuspending, and then ultrasonically homogenizing to obtain PM nanoparticles;

[0020] A glutathione synthesis inhibitor is added to the PM nanoparticles under stirring, the mixture is stirred for reaction, the supernatant is discarded after centrifugation, a glucose solution is added for re-suspending, and then ultrasonic homogenization is performed to obtain dual-drug nanoparticles PMC@HA.

[0021] Furthermore, in the above preparation method, the molar ratio of the zinc nitrate to 2-methylimidazole is 1:20-100; and the molar ratio of the zinc nitrate to copper chloride is 2-4:1.

[0022] Furthermore, the mass ratio of the neutrophil expressed enzyme to the glutathione synthesis inhibitor is as described above. In a specific embodiment of the present invention, the neutrophil expressed enzyme is PPE, the glutathione synthesis inhibitor is CB-839, and the mass ratio of PPE to CB-839 is 3-5:1.

[0023] Furthermore, in the above-mentioned preparation method, the surface of the dual-drug nanoparticles is also modified with hyaluronic acid. In a specific embodiment of the present invention, the process of modifying the surface of the dual-drug nanoparticles with hyaluronic acid is as follows: adding a glutathione synthesis inhibitor to the PM nanoparticles under stirring, stirring the reaction, centrifuging and discarding the supernatant, adding a glucose solution to resuspend, then adding hyaluronic acid, and ultrasonically homogenizing to obtain dual-drug nanoparticles PMC@HA. The mass ratio of zinc nitrate to hyaluronic acid is 2-5:1.

[0024] Furthermore, in the above-mentioned preparation method, the concentration of the glucose solution is 10%, specifically a commercially available 10% glucose injection.

[0025] Furthermore, the stirring reaction conditions are: 600-1000 r / min for 10-60 min (preferably, 800 r / min for 20-30 min); the ultrasonic homogenization conditions are: power 65 W, on for 2 s, off for 3 s, and cycle for 5 min.

[0026] In a specific embodiment of the present invention, the specific preparation process of the dual-drug nanoparticles is as follows: 2-methylimidazole is weighed and dissolved in ultrapure water, PPE is added, and then zinc nitrate solution is added, and magnetic stirring reaction (800r / min reaction 20-30min). Centrifugal supernatant is discarded, glucose solution is resuspended, and then the PPE-wrapped ZIF-8 nanoparticles obtained by ultrasonic homogenization on a probe ultrasonic instrument (power 65W, open 2s, close 3s, cycle 5min), and then copper-based modification is performed, copper chloride solution is added, magnetic stirring reaction is performed, centrifugal supernatant is discarded, glucose is resuspended, and PM nanoparticles are obtained by ultrasonic homogenization under the same conditions. Finally, CB-839 is added under stirring, magnetic stirring reaction is performed, centrifugal supernatant is discarded, glucose is resuspended, hyaluronic acid powder is added, and ultrasonic homogenization is performed to obtain dual-drug nanoparticles PMC@HA.

[0027] The zinc nitrate solution is a 33.45 mg / mL zinc nitrate solution obtained by dissolving zinc nitrate hexahydrate in ultrapure water. The 2-methylimidazole solution is a solution obtained by dissolving 2-methylimidazole in ultrapure water. The copper chloride solution is a 10 mg / mL copper chloride solution obtained by dissolving copper chloride dihydrate in 10% glucose injection. The preparation process of the above solutions is well known to those skilled in the art.

[0028] Preferably, the centrifugation method is 13000 rpm for 5 min.

[0029] In a fourth aspect, the present invention claims protection for the use of the above-mentioned pharmaceutical composition or the above-mentioned dual-drug nanoparticles in the following (1) or (2):

[0030] (1) Application in the preparation of drug combination nano-delivery systems;

[0031] (2) Application in the preparation of anti-tumor drugs.

[0032] The present invention utilizes pH-responsive nano metal organic framework materials. Under mild conditions, PPE and CB-839 in the pharmaceutical composition can coordinate with zinc ions, and the addition of dimethylimidazole can form nanoparticles in situ.

[0033] The dual-drug nanoparticles of the present invention are used as a drug combination nano drug delivery system. The drug combination drug delivery system exhibits good water solubility, biocompatibility and high drug loading capacity, and can achieve anti-tumor immune effects. On the other hand, the nanoparticles can achieve cell endocytosis well, avoiding the extracellular degradation of PPE and CB-839, and its low pH responsiveness can help PPE escape from lysosomes and play a role in killing tumor cells. It lays the foundation for the realization of PPE and CB-839 combined anti-tumor immunotherapy and clinical development.

[0034] The dual-drug nanoparticles PMC@HA modified with hyaluronic acid described in the present invention can form nanoparticles with a particle size of 100.0 to 150.0 nm under neutral to weakly alkaline conditions. Dissolution occurs under acidic conditions to release drugs. The particle size, stability, and biocompatibility of the nanoparticles PMC@HA are all suitable as nanomedicines for tumor immunotherapy. The research of the present invention has shown that PMC@HA can accumulate a large amount of reactive oxygen in the tumor area, significantly downregulate the level of the antioxidant glutathione, destroy its redox balance, and ultimately cause immunogenic cell death of tumor cells, thereby achieving combined anti-tumor immunotherapy.

[0035] The present invention uses copper-doped ZIF-8 as a carrier material to co-load PPE and CB839 for tumor treatment. PPE is an elastase secreted by the pig pancreas, which belongs to the serine protease and is highly homologous to the elastase secreted by human neutrophils. It can cause an increase in the level of mitochondrial reactive oxygen species and play a role in specific tumor cell killing. 2+ The ZIF-8 framework has a dual-responsive release of high GSH and low pH, Cu 2+ It will undergo redox reaction with GSH, consuming GSH and generating Cu + It plays a Fenton-like reaction and can effectively catalyze the decomposition of H2O2 into highly toxic reactive oxygen species under acidic conditions. On the other hand, CB-839 is a glutaminase inhibitor that can inhibit the synthesis of endogenous GSH in tumor cells and upregulate the expression level of CD95 on the surface of tumor cells by blocking glutamine metabolism, thereby maximizing the cell-killing effect of PPE while reducing the level of intracellular GSH. Dual-drug nanoparticles can achieve a synergistic anti-tumor effect of "1+1 is greater than 2" and have good application prospects in the preparation of anti-tumor drugs.

[0036] Beneficial effects of the present invention:

[0037] (1) The pharmaceutical composition containing PPE described in the present invention can be prepared into dual-drug nanoparticles. The nanocarriers encapsulating the two therapeutic drugs can achieve a large amount of cellular endocytosis and can help the drugs escape from lysosomes, release PPE and glutaminase inhibitors in the cytoplasm, and achieve dual-target combined administration of reactive oxygen accumulation and glutathione downregulation, thereby inhibiting tumor growth and metastasis.

[0038] (2) The dual-drug nanoparticles (PMC@HA) of the present invention are based on Zn 2+ As the center, Cu 2+ Partial substitution greatly increased the binding strength and drug loading of PPE and glutaminase inhibitors (such as CB-839) in the nanoparticles, and 2-methylimidazole was used as a ligand to form pH-responsive nanoparticles to avoid extracellular degradation of both drugs.

[0039] (3) The dual-drug nanoparticles described in the present invention improve the properties of free PPE and CB-839, increase their stability, prolong tumor retention time, and enhance therapeutic efficacy.

[0040] (4) The dual-drug nanoparticles described in the present invention can release PPE and CB-839 in response to low pH in intracellular lysosomes, thereby achieving efficient and low-toxic treatment of tumors.

[0041] (5) The hyaluronic acid-modified dual-drug nanoparticles (PMC@HA) described in the present invention can achieve efficient tumor cell surface CD44 targeting by delivering PPE and CB-839 to the tumor area, causing nuclear DNA damage after entry into the cell, and localizing to the mitochondria to produce a large amount of reactive oxygen species with cytotoxic effects, leading to redox imbalance, and ultimately activating immunogenic cell death to achieve tumor killing. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The particle size and potential diagram of PMC@HA in Example 1 of the present invention.

[0043] Figure 2 This is a transmission electron microscope image of PMC@HA in Example 1 of the present invention.

[0044] Figure 3 This is a schematic diagram of the formation of PMC@HA in Example 1 of the present invention.

[0045] Figure 4 This is the X-ray photoelectron spectrum of PMC@HA in Example 1 of the present invention.

[0046] Figure 5 This is the powder X-ray diffraction pattern of PMC@HA in Example 1 of the present invention.

[0047] Figure 6 This is a particle size-time variation diagram of PMC@HA in physiological solution in Example 2 of the present invention, wherein Figures a and b are particle size-time variation diagrams of the nanoparticles in Example 1 of the present invention in glucose injection and culture medium containing 10% serum, respectively.

[0048] Figure 7 3 is a diagram of the in vitro ROS generation of PMC@HA in Example 3 of the present invention, a is the degradation of methylene blue by preparations of different concentrations incubated with the same concentration of H2O2 for 24 hours; b is the degradation of tetramethylbenzidine by the preparations incubated under different pH conditions for 24 hours; c is the degradation of tetramethylbenzidine by the preparations incubated with different concentrations of H2O2 for 24 hours.

[0049] Figure 8This is a diagram showing the depletion of GSH when different concentrations of PMC@HA were incubated with the same concentration of GSH for 24 hours in Example 4 of the present invention.

[0050] Fig. 9 This is a graph showing the cytotoxicity of different preparations to 4T1 cells in Example 5 of the present invention.

[0051] Fig.10 These are confocal images of different preparations of Example 6 of the present invention being taken up by 4T1 cells for 1 h, 2 h, 4 h and 8 h.

[0052] Fig.11 These are confocal images of lysosomal escape of PPE at the subcellular level at 1h, 2h, 4h and 8h in Example 7 of the present invention.

[0053] Fig.12 These are confocal images of the effects of different preparations on mitochondrial membrane potential of 4T1 cells in Example 8 of the present invention. a is a confocal real shot, and b is a quantitative image.

[0054] Fig.13 This is a graph showing the effects of different preparations on ATP levels in 4T1 cells according to Example 9 of the present invention.

[0055] Fig.14 This is a graph showing the effects of different preparations on the HMGB1 level in 4T1 cells according to Example 10 of the present invention. a is a graph showing the extracellular ATP content, and b is a graph showing the intracellular ATP content.

[0056] Fig.15 These are confocal images of the effects of different preparations on lipid peroxidation levels in 4T1 cells according to Example 11 of the present invention.

[0057] Fig.16 This is a graph showing the effects of different preparations on the expression of different proteins in 4T1 cells in Example 12 of the present invention. a is the Glutaminase and CD95 bands, b is the GPX4 band, and c is the H2AX and γH2AX bands.

[0058] Fig.17 This is a graph of the in vivo anti-tumor experiment of different preparations in Example 13 of the present invention, wherein a and b are graphs of the tumor volume and weight after the experiment, respectively. DETAILED DESCRIPTION

[0059] The present invention is further described below by way of examples, but the present invention is not limited to the examples. The experimental methods in the examples of the present invention without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or commodity manufacturers; the reagents without specifying specific sources are conventional reagents available on the market.

[0060] PPE was purchased from MCE with CAS number 39445-21-1.

[0061] CB-839 was purchased from Bidex Pharmaceuticals with a CAS number of 1439399-58-2.

[0062] Example 1 Preparation of PMC@HA Nanoparticles

[0063] Weigh 2-methylimidazole (64.5 mg, molecular weight 82.1 g / mol, molar weight: 0.79 mmol) and dissolve it in 800 μL ultrapure water, add 100 μL of PPE (10 mg / mL), and then add 100 μL of 33.45 mg / mL zinc nitrate solution (3.345 mg zinc nitrate, molecular weight 297.49 g / mol, molar weight: 0.011 mmol), and react with magnetic stirring (800 r / min) for 30 min. The supernatant was discarded after centrifugation, and the nanoparticles were resuspended in 500 μL glucose solution (commercially available 10% glucose injection), and then homogenized on a probe sonicator (power 65W, on 2s, off 3s, cycle 5min), and then copper-based modification was performed, 80 μL of 10 mg / mL copper chloride solution (0.8 mg copper chloride, molecular weight 170.5 g / mol, molar weight: 0.005 mmol) was added, and the reaction was carried out by magnetic stirring (800 r / min) for 30 min, and the supernatant was discarded after centrifugation, and 1 mL of glucose solution (commercially available 10% glucose injection) was resuspended. PM nanoparticles were obtained by ultrasonic homogenization under the same conditions. Finally, 1 mL of CB-839 (0.25 mg / mL) was added under stirring, and the reaction was carried out by magnetic stirring (800 r / min) for 20 min, and the supernatant was discarded after centrifugation, and 500 μL of glucose solution (commercially available 10% glucose injection) was resuspended, and 1 mg of hyaluronic acid powder was added, and ultrasonic homogenization was carried out for 2 min to obtain the final preparation PMC@HA. MC and PMC nanoparticles were prepared in the same way.

[0064] Z nanoparticles: Weigh 2-methylimidazole (64.5 mg, molecular weight 82.1 g / mol, molar weight: 0.79 mmol) and dissolve it in 800 μL ultrapure water, then add 100 μL of 33.45 mg / mL zinc nitrate solution (3.345 mg zinc nitrate, molecular weight 297.49 g / mol, molar weight: 0.011 mmol), stir magnetically (800 r / min) and react for 30 min. Centrifuge and discard the supernatant to obtain Z nanoparticles.

[0065] M nanoparticles: After resuspending the Z nanoparticles, add 80 μL of 10 mg / mL copper chloride solution (0.8 mg copper chloride, molecular weight 170.5 g / mol, molar weight: 0.005 mmol), react with magnetic stirring (800 r / min) for 30 min, and centrifuge and discard the supernatant to obtain M nanoparticles.

[0066] PM nanoparticles: Weigh 2-methylimidazole (64.5 mg, molecular weight 82.1 g / mol, molar weight: 0.79 mmol) and dissolve it in 800 μL ultrapure water, add 100 μL of PPE (10 mg / mL), and then add 100 μL of 33.45 mg / mL zinc nitrate solution (zinc nitrate 3.345 mg, molecular weight 297.49 g / mol, molar weight: 0.011 mmol), and react with magnetic stirring (800 r / min) for 30 minutes. The supernatant was discarded after centrifugation, and the particles were resuspended in 500 μL of glucose solution. The nanoparticles were then homogenized on a probe sonicator (power 65 W, on for 2 s, off for 3 s, and cycle for 5 min), and then copper-based modification was performed by adding 80 μL of 10 mg / mL copper chloride solution (0.8 mg of copper chloride, molecular weight 170.5 g / mol, molar weight: 0.005 mmol), and reacted with magnetic stirring (800 r / min) for 30 min. The supernatant was discarded after centrifugation, and the particles were resuspended in 1 mL of glucose solution. PM nanoparticles were obtained by ultrasonic homogenization under the same conditions.

[0067] PMC nanoparticles: No hyaluronic acid powder was added for ultrasonic homogenization, and the other preparation processes were the same as those of PMC@HA nanoparticles.

[0068] MC nanoparticles: After resuspending the M nanoparticles, add 1 mL of CB-839 (0.25 mg / mL) under stirring, react with magnetic stirring (800 r / min) for 20 min, and centrifuge and discard the supernatant to obtain MC nanoparticles.

[0069] Example 2 Stability of PMC@HA Nanoparticles

[0070] 2 mL of the PMC@HA nanoparticles prepared in Example 1 were dispersed in 4 mL of glucose injection (pH = 7.4) and 1640 culture medium (pH = 7.4) containing 10% fetal bovine serum (FBS), and the particle size and PDI changes of the nanoparticles were monitored by a Malvern laser particle size analyzer. Figure 6 a, b show that the particle size of PMC@HA nanoformulation in glucose injection and 1640 culture medium containing 10% serum was basically stable at different times, respectively, confirming the stability of PMC@HA nanoparticles in a simulated physiological environment.

[0071] Example 3 Investigation of ROS generation in vitro in PMC@HA nanoformulation

[0072] The present invention uses methylene blue (MB) and tetramethylbenzidine (TMB) to detect the generation of ROS in vitro. Under acidic conditions, ·OH produced by H2O2 under the catalysis of copper ions can oxidatively degrade methylene blue, causing methylene blue to fade and the characteristic absorption peak at 590nm-730nm to decrease. In addition, ·OH can catalyze tetramethylbenzidine to generate a blue product with obvious ultraviolet absorption near 652nm.

[0073] A buffer solution containing 100 mM H2O2 at pH = 5.0 was prepared and incubated with 0, 1, 2, 3, and 4 mg / mL of PMC@HA preparations, 1 mL of the sample was centrifuged to collect the supernatant, and the UV absorption of the supernatant at 400 nm-800 nm was scanned by a UV spectrophotometer to measure the degradation of methylene blue induced by ·OH. The degradation results of methylene blue are shown in Figure 7 As shown in a. Buffer solutions containing 100 mM H2O2 at pH = 7.4, 6.5, and 5.0 were prepared and incubated with the preparation for 24 h, respectively. The supernatant was collected by centrifugation, and the degradation of tetramethylbenzidine induced by ·OH was measured by scanning the ultraviolet absorption of the supernatant at 450 nm-800 nm using an ultraviolet spectrophotometer. The degradation results of tetramethylbenzidine are shown in Figure 7 b. 0, 100, 200, 400, 600, 800 mM H2O2 buffer solutions at pH 5.0 were prepared and incubated with the preparations for 24 h, respectively. The supernatants were collected by centrifugation, and the degradation of tetramethylbenzidine induced by ·OH was measured by scanning the ultraviolet absorption of the supernatant at 450 nm-800 nm using an ultraviolet spectrophotometer. The degradation results of tetramethylbenzidine are shown in Figure 7 c. Under acidic conditions, H2O2 basically does not degrade tetramethylbenzidine. Under acidic conditions, the Cu 2+ Reactive oxygen is produced under catalysis to degrade tetramethylbenzidine; different concentrations of H2O2 produce different concentrations of reactive oxygen to degrade tetramethylbenzidine. As the concentration of PMC@HA preparation increases, the concentration of reactive oxygen increases, and the characteristic peak of methylene blue decreases significantly.

[0074] Example 4 Investigation of GSH depletion in vitro by different concentrations of PMC@HA

[0075] The present invention uses 2-nitrobenzoic acid (DTNB) to detect GSSG generated by GSH reaction. The detection principle is that the reduced glutathione (GSH) in the sample to be tested reacts with the chromogenic substrate DTNB to produce stable yellow 2-nitro-5-mercaptobenzoic acid (TNB) and glutathione disulfide (GSSG), and 2-nitro-5-mercaptobenzoic acid is a yellow product with maximum light absorption at a wavelength of 412nm. The absorbance at 412nm is measured by spectrophotometry (spectrophotometer) and compared with the corresponding treated GSH standard to obtain the GSH content of the sample. 0.01mol / L DTNB is dissolved in 0.05mol / L phosphate buffer (pH7.4) to prepare a DTNB standard solution, which is placed away from light and used immediately after preparation. Accurately weigh an appropriate amount of glutathione standard, dissolve it in a volumetric flask with deionized water, mix well, make up to volume to prepare a standard, prepare a pH = 5.0 buffer containing 100mM GSH, incubate with 0, 1.5, 3, 4, 5, 6, 7mg / mL of the preparation for 24h, collect the supernatant by centrifugation, add the same volume of DTNB standard solution, react for 30min, and measure the absorbance by scanning the reaction solution at 412nm with a UV spectrophotometer. The GSH degradation results are shown in Figure 8 As shown. Under acidic conditions, Cu 2+ The reaction with GSH consumed GSH to generate GSSG. With the increase of the concentration of PMC@HA preparation, GSH consumption increased, the color reaction decreased, and the UV absorption decreased. It was confirmed that the prepared PMC@HA nanoparticles had good glutathione peroxidase activity and could consume a large amount of GSH.

[0076] Example 5 Cytotoxic effects of different preparations on 4T1 cells

[0077] The cell 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) experiment was used to evaluate the toxic effects of different preparations (Z, M, PM, PMC, MC, PMC@HA) on 4T1 cells. MTT can react with succinate dehydrogenase in the mitochondria of living cells to generate insoluble blue-purple crystals - formazan; while dead cells cannot produce formazan. Therefore, the amount of formazan generated is proportional to the activity of intracellular enzymes and cell viability, which can reflect the number of cells and the survival status of cells.

[0078] 4T1 cells in the logarithmic growth phase were cultured at 5 × 10 3The cells were seeded at a density of 100 cells / well in a 96-well plate and incubated at 37°C overnight. The supernatant was discarded when the cells adhered to the wall and the confluence reached 80%. Each group of preparations was diluted with fresh culture medium to 10, 20, 30, and 50 μg / mL. Three replicate wells were set up in each group, and control wells and blank wells were set up. 200 μL in each well were incubated with cells at 37°C for 20 hours, and then 20 μL of MTT (concentration of 5 mg / mL) was added to each well, and then incubated in the dark for 4 hours. After discarding the supernatant, 150 μL of DMSO was added to each well and shaken on a shaker for 10 minutes to fully dissolve the formazan. The cell viability was measured using an enzyme reader at 490 nm. The results are as shown in Fig. 9 As shown, the blank vector does not kill 4T1 cells, copper-based modification can enhance its cell killing, and the cytotoxic effect of the PMC@HA group on 4T1 is significantly stronger than that of other groups, confirming the strong tumor killing effect of the nanoformulation. At the same time, it can be seen that the cytotoxicity is concentration-dependent.

[0079] Example 6 Investigation of in vitro uptake capacity of PMC and PMC@HA nanoparticles

[0080] The therapeutic effect of nanomedicine depends on rapid uptake and effective accumulation in cells. Therefore, the cellular uptake of C6-labeled NPs (the preparation method of C6-labeled NPs is the same as that of Example 1, except that CB-839 is replaced by C6) was evaluated by laser scanning confocal microscopy (LSCM).

[0081] 4T1 cells were seeded in confocal culture dishes. When the cells grew to a density of 50%, 500 μL of C6-labeled PMC and PMC@HA resuspended in incomplete culture medium (RPMI 1640, Keygen Biotechnology) were added to each dish. The dishes were incubated in the dark for 1 h, 2 h, 4 h, and 8 h, respectively. The drug-containing culture medium was removed and DAPI staining solution was added to stain for 5 min in the dark. The laser confocal microscope DAPI and FITC channels were turned on and the dishes were observed under the microscope. The confocal results showed (see Fig.10 , Figure a shows the uptake of PMC, and Figure b shows the uptake of PMC@HA). At 1 hour, PMC@HA had been taken up by cells, and the fluorescence intensity increased at 2 hours. The fluorescence intensity was the strongest at 4 hours and reached a plateau, indicating that PMC@HA can be taken up by cells and the uptake behavior is time-dependent. In addition, the fluorescence intensity of the preparation encapsulated with hyaluronic acid is significantly stronger than that of PMC.

[0082] Example 7 Lysosomal escape of PPE in PMC@HA

[0083] 1. Preparation of PPE-FITC-MC@HA

[0084] PPE: FITC (1mg: 150μg): PPE was prepared into 1mg / ml in carbonate buffer at pH 9.0 (carbonate buffer solution: 75.6mg NaHCO3, 10.6mg Na2CO3, 73.6mg NaCl, add water to 10mL); a small amount of DMSO was used to dissolve FITC (20mg / ml freshly prepared); FITC (20mg / ml 7.5μL) was slowly added to PPE solution (1mg / mL1mL) under magnetic stirring, reacted at 1000rpm for 2h in the dark, kept at 4℃ overnight, ultrafiltered and centrifuged at 1500rpm until the lower layer had no fluorescence, and stored at 4 degrees. Then PPE-FITC was used to replace PPE in Example 1 to prepare PPE-FITC-MC@HA.

[0085] 2. 4T1 cells were seeded in confocal culture dishes. When the cells grew to a density of 50%, 500 μL of PPE-FITC-MC@HA resuspended in incomplete culture medium (RPMI 1640 Keygen Biotechnology) was added to each dish. After incubation in the dark for 1h, 2h, 4h and 8h, the drug-containing culture medium was removed. DAPI staining solution was added for staining in the dark for 5min, and Lyso Tracker Red lysosomal probe was used for staining in the dark for 30min. The DAPI, FITC and Lyso Tracker Red channels of the laser confocal microscope were turned on and observed under the microscope. By observing the distribution of fluorescent signals in the cells under a fluorescence microscope, it can be determined whether the substance to be detected can escape from the lysosome. If PPE-FITC and lysosomal marker dyes are completely colocalized, it means that PPE-FITC is mainly present in lysosomes and has not escaped from lysosomes; if the fluorescence signal of PPE-FITC is observed to be distributed in the cytoplasm or nucleus outside the lysosomes and does not completely overlap with the distribution of lysosomal marker dyes, it indicates that PPE-FITC has escaped from lysosomes. Fig.11 As shown, at 1 h, the green fluorescence of PPE-FITC and the red fluorescence of lysosomes completely overlapped, showing orange fluorescence. At 2 h, PPE-FITC began to escape from lysosomes, and then the green fluorescence increased with time.

[0086] Example 8 Effects of different preparations on mitochondrial membrane potential of 4T1 cells

[0087] The large accumulation of mitochondrial reactive oxygen species can cause mitochondrial dysfunction to a certain extent, resulting in a decrease in mitochondrial membrane potential. JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential (ΔΨm). When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers (J-aggregates), which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix. At this time, JC-1 is a monomer and can produce green fluorescence. The relative ratio of red and green fluorescence is often used to measure the proportion of mitochondrial depolarization. The decrease in mitochondrial membrane potential is a landmark event in the early stage of cell apoptosis.

[0088] The mitochondrial status of tumor cells was detected using the Beyotime 1,1',3,3'-tetraethyl-iminodicarbocyanine (JC-1) probe. 4T1 cells were seeded on 35 mm CLSM dishes (10 cells per dish). 5 cells, 1640 culture medium), and after 24 h of adherent culture, the original culture medium was replaced with a culture medium containing the material (10 μg mL -1 ). After incubation with the material for 24 hours, the culture medium was removed and the cells were carefully washed with PBS. After treating the cells with the pre-prepared JC-1 working solution in the dark for half an hour, the working solution in the culture dish was replaced with an equal volume of PBS. The cell state was observed and pictures were taken using a laser confocal microscope (the red fluorescence excitation wavelength and emission wavelength were 585nm and 590nm, respectively; the green fluorescence excitation wavelength and emission wavelength were 514nm and 525nm, respectively), and the proportion of red and green fluorescence was analyzed.

[0089] Under normal circumstances, the mitochondrial membrane has a high potential, and JC-1 will form aggregates under this condition and emit red fluorescence under the irradiation of excitation light. In cells that are about to undergo apoptosis (late stage of apoptosis), the mitochondria will break and the membrane potential will decrease. In this case, JC-1 that enters the cell cannot aggregate and exists in the cell as a monomer. The dye monomer will emit green fluorescence in the presence of excitation light. The killing effect of the material on tumor cells was further verified by staining 4T1 cells co-incubated with the material with JC-1. Fig.12 a As can be seen, the Control group showed red fluorescence, while in the PMC group and PMC@HA group, green fluorescence was dominant, and some of the MC and PM groups were stained yellow, which was the result of the coexistence of red and green fluorescence, indicating that there were some normal mitochondria in the cells, but also a large number of damaged and broken mitochondria. By analyzing the red and green fluorescence ratios of each group ( Fig.12b), PMC@HA has a higher proportion of green fluorescence than other groups, indicating that the material causes the most serious damage to mitochondria, that is, PMC@HA has a stronger anti-tumor effect.

[0090] Example 9 Effects of different preparations on ATP levels in 4T1 cells

[0091] 4T1 cells in the logarithmic growth phase were cultured at 5×10 6 The cells were inoculated at a density of 100 μl / well in a 6-well plate and cultured until the cell confluence reached 80%. The nanoparticles prepared in Example 1 were added to each well, and three replicate wells were set up with 200 μL in each well. After 24 hours of incubation, the cells were collected and centrifuged. The supernatant was tested and the cells were lysed using RIPA cell lysis buffer (Sevier, China). After lysis, centrifuge at 4°C and 12000g for 10 minutes, take the supernatant, and in order to eliminate the error caused by the difference in protein amount during sample preparation, the protein concentration was determined according to the instructions of the BCA protein detection kit. The ATP concentration was determined according to the instructions of the ATP detection kit, and the results are as follows: Fig.13 As shown, the release of ATP from dying cells into the extracellular space constitutes one of the main signs of ICD. It can be seen that the free mixed group can cause ATP to be released into the extracellular space. The effect of the final preparation group is significantly better than that of the free mixed group, proving that the preparation has a more significant effect on ICD, and has obvious advantages in inducing the anti-tumor pathway of ICD through copper death.

[0092] Example 10 Effects of different preparations on HMGB1 levels in 4T1 cells

[0093] 4T1 cells in the logarithmic growth phase were cultured at 5×10 6 The cells were inoculated at a density of cells / well in a 6-well plate and cultured until the cell confluence reached 80%. The nanoparticles prepared in Example 1 were added to each well, and three replicate wells were set up with 200 μL in each well. After 24 hours of incubation, the cell sample was centrifuged to take the supernatant (500×g, 5 minutes) and the cells were lysed using RIPA cell lysis buffer (Sevier, China). After lysis, centrifuge at 4°C and 12000g for 10 minutes, take the supernatant, and in order to eliminate the error caused by the difference in protein amount during sample preparation, the protein concentration was determined according to the instructions of the BCA protein detection kit. The HMGB1 concentration of the cell sample supernatant was determined according to the instructions of the HMGB1 detection kit, and the results are as shown below. Fig.14 As shown, the release of high mobility group protein 1 (HMBG1) is also one of the main signs of ICD. It can be seen that the free mixed group can cause the release of HMBG1 to the extracellular space. The effect of the final preparation group is significantly better than that of the free mixed group, indicating that the combination of the two drugs to form nanoparticles significantly enhanced the anti-tumor effect.

[0094] Example 11 Effects of different preparations on lipid peroxidation levels in 4T1 cells

[0095] 4T1 cells in the logarithmic growth phase were seeded in a 24-well plate at a density of 10,000 cells / well and cultured until the cell confluence reached 80%. Each group of nanoparticles and free drug physical mixtures prepared in Example 1 were added to each well, with three replicate wells in each group, 1 mL in each well, and incubated for 24 hours. The culture medium was removed, and the cells were washed twice with PBS, incubated in a serum-free medium containing 2 μM C11BODIPY 581 / 591 in the dark at 37°C for 30 minutes, washed twice with PBS, incubated in a medium containing 2 μM DAPI in the dark at 37°C for 10 minutes, washed twice with PBS, and finally, fluorescence imaging was performed using a confocal laser scanning microscope. The results are shown in FIG. Fig.15 As shown. The weaker the fluorescence intensity of the reduced dye in C11 BODIPY 581 / 591, the stronger the degree of cellular lipid peroxidation. The free drug physical mixing group had little effect on 4T1 cell lipid peroxidation, while the PMC@HA group significantly increased the degree of 4T1 cell lipid peroxidation. This was because it produced ROS with stronger oxidative activity under the catalysis of monovalent copper ions. ROS further oxidized lipids in cells, and free radicals produced by mitochondrial dysfunction caused by PPE could also cause lipid peroxidation.

[0096] Example 12 Effects of different preparations on the expression of different proteins in 4T1 cells

[0097] Western blot analysis: Western blot analysis was used to determine the effects of different preparations on the expression of proteins in the Glutaminase, CD95, GPX4, H2AX, and γH2AX pathways. Proteins were extracted from cells using RIPA cell lysis buffer (Solerbo, China). Protein concentrations were determined according to the instructions of the BCA protein assay kit. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electrophoresed the relevant equal amounts of proteins, and after the electrophoresis, the proteins were transferred to a PVDF membrane. A blocking solution containing skim milk powder was applied to the membrane, and then incubated with diluted anti-GPX4 primary antibody overnight at 4°C. The membrane was quickly and repeatedly washed and incubated with diluted corresponding secondary antibodies (1:5000) at room temperature for 30 minutes. ECL chemiluminescence detection kit was used for color development, and the rest of the Western blot analysis was the same as above. CB-839 is a small molecule glutaminase inhibitor that can inhibit glutaminase in cells. The combination of copper-based carrier and CB-839 can significantly downregulate intracellular glutathione levels, thereby causing the upregulation of CD95 targets. Fig.16 a It can be seen that the Glutaminase protein in the PMC@HA group was significantly downregulated, and the CD95 protein was significantly upregulated. Fig.16Glutathione peroxidase 4 (GPX4) shown in b is a selenoprotein that can specifically catalyze glutathione to convert lipid peroxides into lipopolysaccharides. It plays an important role in regulating cell copper death. Copper-based modified carriers can significantly downregulate intracellular GPX4 levels. The main mechanism of PPE is to act on the CD95 target, which then causes nuclear DNA damage, leading to tumor killing. Histone H2AX plays an important role in nucleosome formation, chromatin remodeling and DNA repair. Once the double-stranded DNA is broken, it is phosphorylated at the Ser139 site to become γH2AX. γH2AX can mark the site of double-strand breaks and recruit cell cycle checkpoints and DNA repair factors to the damaged site. Therefore, the two proteins H2AX and γH2AX were selected to determine the extent of nuclear DNA damage caused by nanoparticles. Fig.16 As shown in c, the γH2AX / H2AX ratio of the PMC@HA group was the highest, and the degree of DNA damage was the strongest.

[0098] Example 17 In vivo antitumor experiments of different preparations

[0099] The 4T1 cell suspension (1x106 cells / 100 μL) was inoculated on the right back of female Balb / C mice. When the tumor volume grew to 50 mm 3 At the same time, tumor-bearing mice were randomly divided into groups of five mice each and given physiological saline, free drug mixture, MC, PM, PMC and PMC@HA prepared in Example 1, respectively. The drugs were given once every two days for six consecutive times at a dose of 5 mg / kg. Fig.17 Compared with the control group, the PMC@HA group can significantly inhibit tumor growth and has a stronger inhibitory effect on tumor growth by inducing cell copper death in tumor cells; the nanoformulation has the strongest inhibitory effect on tumor growth by combining chemodynamic therapy and immunotherapy.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a neutrophil-expressed enzyme and a glutathione synthesis inhibitor, wherein the neutrophil-expressed enzyme is at least one of neutrophil elastase and porcine pancreatic elastase; and the glutathione synthesis inhibitor is at least one of BSO, V-9302 and CB-839.

2. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of the neutrophil expression enzyme to the glutathione synthesis inhibitor is 1:100 to 100:

1.

3. A dual-drug nanoparticle, characterized in that: The dual-drug nanoparticles are nano-composite materials Cu / ZIF-8 obtained by using a copper-based modified ZIF-8 carrier as a carrier, and the carrier contains a neutrophil expression enzyme, and the carrier adsorbs a glutathione synthesis inhibitor to form a nano-drug; the neutrophil expression enzyme is at least one of neutrophil elastase and porcine pancreatic elastase; the glutathione synthesis inhibitor is at least one of BSO, V-9302 and CB-839.

4. The dual-drug nanoparticle according to claim 3, characterized in that: The surface of the dual-drug nanoparticles is also modified with hyaluronic acid.

5. The dual-drug nanoparticle according to claim 3, characterized in that: The Zn in the nanocomposite Cu / ZIF-8 2+ With Cu 2+ The molar ratio of Zn in the ZIF-8 carrier is 2-4:1; 2+ The molar ratio of 2-methylimidazole is 1:20-100.

6. The method for preparing the dual-drug nanoparticles according to claim 3, characterized in that: The preparation method is: The neutrophil-expressed enzyme and zinc nitrate solution were added to the 2-methylimidazole solution, stirred for reaction, centrifuged and the supernatant was discarded, glucose solution was added for resuspending, and then ultrasonic homogenization was performed to obtain ZIF-8 nanoparticles encapsulating the neutrophil-expressed enzyme; Adding copper chloride solution to the ZIF-8 nanoparticles for copper-based modification, stirring for reaction, centrifuging and discarding the supernatant, adding glucose solution for resuspending, and then ultrasonically homogenizing to obtain PM nanoparticles; A glutathione synthesis inhibitor is added to the PM nanoparticles under stirring, the mixture is stirred for reaction, the supernatant is discarded after centrifugation, a glucose solution is added for resuspending, and then ultrasonic homogenization is performed to obtain dual-drug nanoparticles PMC@HA.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the zinc nitrate to 2-methylimidazole is 1:20-100; the molar ratio of the zinc nitrate to copper chloride is 2-4:

1.

8. The preparation method according to claim 6, characterized in that: The surface of the dual-drug nanoparticles is also modified with hyaluronic acid.

9. The preparation method according to claim 6, characterized in that: The mass ratio of the neutrophil expression enzyme to the glutathione synthesis inhibitor is 1:100 to 100:

1.

10. Use of the pharmaceutical composition according to claim 1 or 2, or the dual-drug nanoparticles according to any one of claims 3 to 5 in the following (1) or (2), (1) Application in the preparation of drug combination nano-delivery systems; (2) Application in the preparation of anti-tumor drugs.

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