A nanoparticle for multidimensionally reducing cisplatin nephrotoxicity, its preparation method and application

Abstract

The present invention provides a nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity, its preparation method and application. The nanoparticle is formed by coupling tea polyphenols with carboxylated cisplatin and self-polymerizing through catalytic oxidation by divalent metal manganese ions in a weakly alkaline environment with a pH of 8.0. Its preparation method is simple. First, cisplatin is oxidized by an oxidant and then carboxylated, and then dissolved in a weakly alkaline buffer solution with tea polyphenols, stirred evenly at room temperature, then a metal manganese ion solution is added for catalysis, and magnetic stirring is carried out under a constant temperature water bath, and then Pt-TPNs nanoparticles are obtained through centrifugation and washing. The nanoparticles reduce the damage of cisplatin to the kidney through multiple dimensions such as scavenging ROS, blocking inflammatory reactions, and inhibiting pyroptosis, and have tumor targeting, which can provide a basis and idea for the treatment of tumors. It is a potential multi-target and highly efficient nano-drug for anti-tumor treatment; its composition is controllable, and it has the excellent characteristics of being cheap and easily available, simple to prepare, mild in conditions, and can be prepared in large quantities, which is convenient for popularization and application.

Description

Technical Field

[0001] The present invention relates to the fields of nanomaterials and nano-biomedicine, and specifically relates to a method for constructing and applying a nanopreparation for multi-dimensionally reducing cisplatin nephrotoxicity. Background Art

[0002] Cisplatin is a first-line drug for many cancer patients. It exerts its therapeutic effect by interacting with the bases at the N7 site of DNA in cancer cells, causing DNA cross-linking and thus inducing cell death. However, there are also many problems in the clinical application of cisplatin, such as nephrotoxicity, ototoxicity, myelosuppression, etc. Among them, nephrotoxicity is the dose-limiting factor of cisplatin, severely restricting the clinical application of cisplatin. At present, the main method to alleviate cisplatin-induced nephrotoxicity clinically is hydration therapy, which not only affects the efficacy of cisplatin but also has poor effects. Therefore, how to protect the kidneys during cisplatin chemotherapy is a key problem that needs to be solved at present. Existing studies have shown that cisplatin nephrotoxicity comes from many aspects, among which pyroptosis, oxidative stress and inflammation are the main factors. In view of different damage mechanisms, scientific researchers have tried various protective measures, such as scavenging ROS, blocking inflammatory responses, protecting mitochondrial function, inhibiting apoptosis, etc., and certain effects have been achieved. However, due to the complex mechanism of cisplatin nephrotoxicity, intervening only from a certain angle may be compensated through other pathways. Therefore, it is urgent to construct a new treatment strategy for multi-directional synchronous blocking for the above mechanisms. Summary of the Invention

[0003] To solve the above technical problems, the present invention proposes a nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity, its preparation method and application. The purpose is to catalytically oxidize and self-polymerize Pt-EGCG to form nanoparticles in a weakly alkaline environment through a specific catalyst. The nanoparticles can alleviate oxidative stress, inflammation and inhibit pyroptosis, thereby reducing the nephrotoxicity of cisplatin; the nanoparticles have high stability and have an anti-tumor effect.

[0004] To achieve the above object, the present invention first provides a nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity, including tea polyphenols and cisplatin. The nanoparticle is formed by catalytic oxidation and self-polymerization of tea polyphenols and cisplatin after bonding in a weak base environment through Mn 2+ catalysis.

[0005] Preferably, the cisplatin is carboxylated cisplatin, and the carboxylated cisplatin is bonded to the tea polyphenols through a carboxyl group.

[0006] Preferably, the Mn 2+ catalyst is MnCl2·4H2O, and the weak alkalinity is pH 7.4 - 8.5.

[0007] Based on a general inventive concept, the present invention also provides a preparation method of the nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity, including the following steps:

[0008] S1. Oxidize cisplatin with H2O2, cool it after vigorous stirring, wash it, and then freeze-dry to obtain the initial product Pt(OH)2.

[0009] S2. Dissolve Pt(OH)2 and succinic anhydride in DMSO, stir vigorously and then freeze-dry, then inject acetone to precipitate crystals at low temperature, and dry the crystals to obtain carboxylated cisplatin Pt(COOH)2.

[0010] S3. Mix Pt(COOH)2, EDC · HCl, and DMAP, add DMSO and stir in a water bath, then add EGCG and continue stirring to obtain tea polyphenols-bonded cisplatin Pt-EGCG.

[0011] S4. Add the Pt-EGCG reaction solution to a weakly alkaline buffer solution, add Mn 2+ and PVP while stirring, centrifuge after a water bath, and perform ultrasonic treatment to obtain nanoparticles Pt-TPNs with multi-dimensional reduction of cisplatin nephrotoxicity.

[0012] Preferably, in step S1, the concentration of H2O2 is 30%; the temperature of the stirring is 45 °C, the stirring time is 24 h; the cooling temperature is 4 °C, and the cooling time is 1 d.

[0013] Preferably, in step S2, the molar ratio of Pt(OH)2 to succinic anhydride is 1:3; the temperature of the stirring is 70 °C, the stirring time is 24 h; the crystallization temperature is -20 °C; the drying temperature is 50 °C, and the drying time is 3 h.

[0014] Preferably, in step S3, the molar ratio of Pt(COOH)2 to EGCG is 1:1 - 2; the water bath temperature is 27 °C, the water bath stirring time is 3 h; the continued stirring time is 24 h.

[0015] Preferably, in step S4, the molar ratio of Pt-EGCG to Mn 2+ is 10:1 - 1:10.; the buffer solution is HEPES buffer solution; the final concentration of the HEPES buffer solution is 10 mM; the concentration of PVP is 20%; the water bath temperature is 27 °C, the water bath stirring time is 3 h; the centrifugation speed is 16000 rpm, the centrifugation time is 10 min; the power of the ultrasonic probe is 40 w, the ultrasonic time is 3 s, and the number of ultrasonic times is 2.

[0016] Based on a general inventive concept, the present invention also provides an application of nanoparticles with multi-dimensional reduction of cisplatin nephrotoxicity in the preparation of cisplatin drugs with low nephrotoxicity.

[0017] Based on a general inventive concept, the present invention also provides an application of a nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity in the preparation of an anti-tumor drug.

[0018] The working mechanism of the nanoparticle prepared by the present invention in alleviating cisplatin-induced nephrotoxicity is as follows:

[0019] Using the oxidizing property of H2O2 to oxidize cisplatin, reacting with succinic anhydride to generate carboxylated cisplatin, and coupling the carboxylated cisplatin with tea polyphenol (EGCG) in a chemical bonding manner. Under weak alkaline conditions, through the oxidation and self-polymerization catalyzed by Mn 2+ nanoparticles Pt-TPNs carrying cisplatin are formed by self-polymerization. In the Pt-TPNs nanoparticles of the present invention, Mn 2+ catalyzes the reaction of tea polyphenol Pt-EGCG bonded with cisplatin in a weak alkaline environment. Among them, EGCG is rapidly oxidized under catalysis, and the catechol and pyrogallol units are converted into corresponding highly active semiquinones and quinones. Through a series of nucleophilic addition reactions between quinones and semiquinones, or through the coupling reaction of semiquinone radicals, through the oxidative coupling polymerization reaction between EGCG molecules, the tea polyphenol Pt-EGCG bonded with cisplatin is oxidized and polymerized to form Pt-TPNs nanoparticles; the nanoparticles have a spherical structure, and the particle size has no significant change within 48 h, and have good colloidal stability. The Pt-TPNs nanoparticles show excellent anti-tumor effects in vivo and in vitro, and alleviate the lesions and damages of cisplatin to kidney tissues or kidney cells from multiple angles such as relieving oxidative stress, inflammation, and inhibiting pyroptosis.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The Pt-TPNs nanoparticles provided by the present invention are formed by coupling tea polyphenol (EGCG) with carboxylated cisplatin and oxidizing and self-polymerizing under the catalysis of divalent manganese ions in a weak alkaline environment. Among them, manganese ions only catalyze the oxidative self-polymerization of Pt-EGCG and will not complex with Pt-EGCG and enter the nanoparticle structure. In this way, the nanoparticles formed by oxidative self-polymerization have a particle size of 180 - 260 nm, good dispersibility and water solubility, simple and stable structure, and high biocompatibility.

[0022] 2. The present invention provides a preparation method of Pt-TPNs nanoparticles. The preparation process is simple and controllable, the reagents are simple, and the metal manganese ions only play a catalytic role. The composition of the nanoparticles is controllable. The self-polymerized nanoparticles have the excellent characteristics of low cost, easy availability, simple preparation, mild conditions, and can be prepared in large quantities, which is convenient for popularization and application.

[0023] 3. The present invention provides an innovative application of Pt-TPNs nanoparticles in reducing the renal injury caused by cisplatin preparations. Compared with the single perspective of traditional renal protection means, the present invention reduces the damage of cisplatin to the kidneys through multiple dimensions such as scavenging ROS, blocking inflammatory responses, and inhibiting pyroptosis, and has tumor targeting, which can provide a basis and idea for tumor treatment. It is a potential multi-target and highly efficient nano-drug for anti-tumor treatment.

[0024] 4. In addition to scavenging free radicals and blocking the pyroptosis program by itself, the nanoparticles provided by the present invention can also serve as carriers for other drugs, showing great potential in the preparation of tumor treatment drugs.

[0025] 5. The present invention creatively discovers that the Pt-TPNs nanoparticles have excellent activities in spectrally scavenging reactive oxygen species and reactive nitrogen species, and can be used for the preparation of drugs for various inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 It is the transmission electron micrograph of Pt-TPNs nanoparticles in Experimental Example 1 of the present invention;

[0028] Figure 2 It is the particle size distribution diagram of Pt-TPNs nanoparticles in Experimental Example 1 of the present invention;

[0029] Figure 3 It is the graph of the change of particle size of Pt-TPNs nanoparticles with time in pure water, PBS, HEPES, and 10% FBS complete medium in Experimental Example 1 of the present invention;

[0030] Figure 4 It is for Pt-TPNs nanoparticles on tumor cells and renal proximal tubular cells in Experimental Example 2 of the present invention, Figure 4 Among them, a and b respectively refer to the cytotoxicity test diagram of 143B tumor cells and the cytotoxicity test diagram of HK-2 renal proximal tubular cells;

[0031] Figure 5 It is the laser confocal imaging diagram of tumor cell uptake of Pt-TPNs nanoparticles in Experimental Example 3 of the present invention;

[0032] Figure 6 It is the laser confocal imaging diagram of lysosomal escape of Pt-TPNs nanoparticles in Experimental Example 4 of the present invention;

[0033] Figure 7 This is the effect diagram of Pt-TPNs nanoparticles on free radical scavenging in Experimental Example 5 of the present invention; Figure 7 In it, A, B, and C respectively refer to the fluorescence images of cells loaded with DCFH-DA, DHE, and DAF-DA fluorescence probes under different treatments, representing the total ROS, ·O 2 - and ·NO levels in cells, scale bar = 100 μm;

[0034] Figure 8 This is the effect diagram of Pt-TPNs nanoparticles on inhibiting pyroptosis in Experimental Example 6 of the present invention;

[0035] Figure 9 This is the mechanism diagram of Pt-TPNs nanoparticles on inhibiting pyroptosis in Experimental Example 6 of the present invention. Figure 9 (A) represents the experimental diagram of the effect of TPNs on the cleavage of GSDME and Caspase-3 proteins. Figure 9 (B) represents the experimental result diagram of TPNs inhibiting the oligomerization of the N-terminus of GSDME on the cell membrane;

[0036] Figure 10 This is the pharmacokinetic study diagram of Pt-TPNs nanoparticles and cisplatin in Experimental Example 7 of the present invention. Figure 10 In it, a, b, and c respectively refer to the change in the concentration of Pt drug in the blood within 0 - 24 h after administration, the tissue distribution of Pt 6 h after administration, and the tissue distribution of Pt 24 h after administration;

[0037] Figure 11 This is the characterization diagram of Pt-TPNs nanoparticles relieving renal toxicity in vivo in Experimental Example 8 of the present invention; Figure 11 In it, a, b, c, d, e, and f respectively refer to the content of renal injury factor Cre, the content of renal injury factor BUN, the kidney index, HE staining of renal tissue, renal injury score, and immunohistochemistry of renal tissue;

[0038] Figure 12 This is the research diagram of Pt-TPNs nanoparticles relieving oxidative stress in vivo in Experimental Example 9 of the present invention; Figure 12 In it, a, b, c, and d respectively refer to the content of oxidative stress-related factor GSH, the content of oxidative stress-related factor CAT, the content of lipid oxidation product MDA, and the expression of Nitro tyrosine and 8-OHdG proteins in renal tissue;

[0039] Figure 13 This is the research diagram of Pt-TPNs nanoparticles scavenging inflammatory factors in vivo in Experimental Example 10 of the present invention; Figure 13 In it, a, b, c, and d respectively refer to the content of inflammatory factor IL-1α, the content of inflammatory factor IL-1β, the content of inflammatory factor TNF-α, and the content of inflammatory factor IL-6;

[0040] Figure 14 This is the research figure of Pt-TPNs nanoparticles inhibiting pyroptosis in vivo in Experimental Example 11 of the present invention;

[0041] Figure 15 This is the research figure of Pt-TPNs nanoparticles inhibiting tumor growth in vivo in Experimental Example 12 of the present invention;

[0042] Figure 16 This is the evaluation figure of the in vivo safety of Pt-TPNs nanoparticles in Experimental Example 13 of the present invention. Detailed implementation manners

[0043] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0044] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0045] If not specifically specified, the technical means used in the examples are conventional means well-known to those skilled in the art; if not specifically specified, the reagents used in the examples are all commercially available.

[0046] The percentage "%" involved in the present invention, if not specifically stated, refers to the mass percentage; but for the percentage of the solution, unless otherwise specified, it refers to the number of grams of solute contained in 100 ml of the solution.

[0047] The parts by weight described in the present invention can be well-known weight units in the art such as μg, mg, g, kg, etc., or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0048] In the following examples, the detailed information of the instruments and manufacturers used is shown in Table 1:

[0049] Table 1 Main instrument names and manufacturers

[0050] Instrument Name Manufacturer CP225D Electronic Balance Sartorius Company, Germany BP224S Electronic Balance Sartorius Company, Germany XW-80A Vortex Mixer Shanghai Qingpu Huxi Analytical Instrument Factory Handheld Centrifuge Scilogex Company, USA DF-101S Constant Temperature Heating Magnetic Stirrer Gongyi Yuhua Instrument Co., Ltd. UV-2600 UV Spectrophotometer Shimadzu Corporation, Japan TGL16M Low Temperature High Speed Centrifuge Changsha Yingtai Instrument Co., Ltd. TD4A Desktop Low Speed Centrifuge Changsha Yingtai Instrument Co., Ltd. SHA-B Water Bath Thermostatic Oscillator Changzhou Aohua Instrument Co., Ltd. MIN4-UVF Pure Water Machine Hunan Kelton Water Service Co., Ltd. UV-2600 UV Spectrophotometer Shimadzu Corporation, Japan Infinite M200PRO Multifunctional Microplate Reader TECAN Company, Austria Nano-ZS90 Particle Size Analyzer Malvern Instruments Limited, UK Tecnai G2 F20 Transmission Electron Microscope FEI Company, USA 1×70 Inverted Fluorescence Microscope Olympus Corporation, Japan Forma Series II CO2 Incubator Thermo Fisher Scientific, USA SW-CJ-2FD Vertical Laminar Flow Hood Suzhou Purification Equipment Co., Ltd. DSX-30L Autoclave Shanghai Shen'an Medical Instrument Factory Refrigerator Haier Medical Refrigerator, China Freezer Haier Medical Low Temperature Preservation Refrigerator, China 7700X ICP-MS Agilent Technologies, Japan CEBO-6 Small Tissue Homogenizer Shanghai Cebo Biotechnology Development Center YT-6C Biological Tissue Spreader and Oven Xiaogan Yaguang Medical Electronics Co., Ltd.

[0051] In the following examples, the names and manufacturers of the main reagents used are shown in Table 2:

[0052] Table 2 Main reagent names and manufacturers

[0053]

[0054]

[0055] Example 1

[0056] Preparation of nanoparticles (Pt-TPNs) with multi-dimensional reduction of cisplatin nephrotoxicity

[0057] S1. Weigh 500 mg of cisplatin precisely into a round-bottom flask, add 20 mL of H2O2 (30%), seal it with a sealing film, puncture holes with a syringe needle, place it in an oil bath, stir vigorously at 45 °C for 24 h, then place the round-bottom flask in a 4 °C cooler for 1 day, wash it several times with water, and freeze-dry and weigh: 491.97 mg.

[0058] S2. Weigh 333 mg of Pt(OH)2 (1 mmol) and 302.1 mg of succinic anhydride (3 mmol) precisely, place them in a 25 mL round-bottom flask, add 3 mL of DMSO, place it in an oil bath, stir vigorously at 70 °C for 24 h, then freeze-dry DMSO, inject 20 mL of acetone, crystallize at -20 °C, wash with acetone several times, dry the crystals in a vacuum drying oven at 50 °C for 3 h, and weigh 510 mg.

[0059] S3. Weigh 53.4 mg of Pt(COOH)2, 38.28 mg of EDC HCl, and 18.32 mg of DMAP into a 10 mL EP tube, add 3 mL of DMSO, stir in a water bath at 27 °C for 3 h, add 137.52 mg of EGCG, and stir for 24 h to obtain Pt-EGCG.

[0060] S4. Add 60 μL of Pt-EGCG reaction solution to 2.4 mL of HEPES (pH 8.0, 10 mM) buffer solution, add 48 μL of MnCl2·4H2O and 20 μL of 20% PVP under magnetic stirring, stir in a water bath at 27 °C for 3 h, centrifuge at 16000 rpm for 10 min, wash twice with water, and ultrasonicate with a probe at 40 w for 3 s, 2 times to obtain the nano-formulation Pt-TPNs.

[0061] Experimental Example 1

[0062] Investigate the morphology, encapsulation of cisplatin, particle size and stability of Pt-TPNs

[0063] I. Morphology: Drop the sample onto a 400-mesh copper grid covered with a carbon film, place it in a desiccator, and observe it under a transmission electron microscope Titan G2-F20 after it dries naturally. The transmission electron microscope image is as Figure 1 shown, indicating that the nanoparticles are spherical in structure.

[0064] II. Encapsulation of cisplatin by the nano-carrier: Prepare the nano-complex according to the method of Example 1, and perform ICP determination on the nano-complex to verify the encapsulation of cisplatin.

[0065] The drug loading rate and encapsulation rate are shown in Table 3:

[0066] Table 3 Drug loading and encapsulation efficiency of Pt-TPNs nanoparticles

[0067]

[0068] The drug loading of Pt-TPNs for cisplatin was 5.3%, and the encapsulation efficiency was as high as 27.2%.

[0069] III. Particle size: The particle size of Pt-TPNs was detected. The measurement method was as follows: The sample solution was placed in a Malvern Nano ZS instrument, and the particle size was detected by dynamic light laser scattering method. The temperature of the measuring cell was set at 25 °C, and three parallel operations were performed for each sample. The results are as Figure 2 shown. The curve shown is the particle size distribution of the nanoformulation. It can be seen that its particle size is uniform, and the particle size is 180 - 260 nm.

[0070] IV. Stability detection: Pt-TPNs were placed in PBS, HEPES, 10% FBS complete medium, and pure water at 37 °C, and their particle sizes were measured at different time points. Figure 3 is the particle size change diagram in PBS, 10% FBS complete medium, and pure water. It can be seen from the figure that: The particle size of Pt-TPNs did not change significantly within 48 h, indicating that the nano-carrier has good colloidal stability.

[0071] Experimental Example 2

[0072] Investigate the anti-tumor efficacy and characteristics of reducing renal toxicity of cisplatin and Pt-TPNs at the cellular level

[0073] HK-2 cells (1×10 4 / well) and 143B cells (5×10 3 / well) were seeded in 96-well plates respectively and adhered for 24 h. The TPNs group (blank control, 5, 10, 20, 30, 40, 50 L, 100 μg / mL), cisplatin group (blank control, 2, 5, 10, 15, 20, 30, 45 μM, calculated by Pt content), and Pt-TPNs group (the concentration was set the same as that of cisplatin) were set. After 48 h of drug treatment, the supernatant was discarded, washed twice with PBS, 100 μL of 0.5 mg / mL MTT was added, incubated in an incubator for 4 h, the supernatant was discarded, 150 μL of DMSO was added, shaken on a shaker at 37 °C for 20 min, and the absorbance was measured at 490 nm.

[0074] The results are as Figure 4As shown in the figure, in cancer cells (143B cells), cisplatin has a significant effect on the cell viability of cancer cells. As the concentration of cisplatin increases, the viability of 143B cells gradually decreases, indicating that the higher the concentration of cisplatin, the more it promotes the apoptosis of cancer cells. The TPNs group without cisplatin has no significant effect on the viability of cancer cells, and the cell viability remains between 80% - 100%. However, in the Pt-TPNs group of the present invention, as the Pt concentration gradually increases, the inhibitory effect on cancer cells becomes more obvious. During the process of the Pt concentration increasing from 40 μM to 100 μM, the cell viability decreases from 80% to 20%.

[0075] In human renal tubular epithelial HK-2 cells, the higher the concentration of cisplatin, the more obvious the promotion of apoptosis of normal human renal tubular epithelial cells. However, the Pt-TPNs prepared in the present invention do not promote the apoptosis of renal tubular epithelial cells as the Pt concentration increases.

[0076] This experimental example fully demonstrates that the Pt-TPNs of the present invention can effectively inhibit cancer cells while alleviating the damage of high-concentration Pt to the kidney.

[0077] Experimental Example 3

[0078] Investigate the characteristics of tumor cell uptake of Pt-TPNs

[0079] Seed 143B cells at a density of 1×10 5 per well in a confocal dish and allow them to adhere and grow for 24 h. Set up blank control, FAM-siRNA, and Pt-TPNs / FAM-siRNA groups (the amount of FAM-siRNA is the same as that encapsulated in the nanoparticles, both are 500 nM. The preparation method is as follows: in a 2 mL EP tube, add 240 μL of HEPES (8.0, 10 mM) buffer, 6 μL of Pt(COOH)2-EGCG reaction solution, and 9.6 μL of 100 μM FAM-siRNA, stir magnetically for crosslinking for 30 min, then add 4.8 μL of MnCl2·4H2O and 20 μL of 20% PVP, incubate in a water bath at 27 °C for 2.5 h, centrifuge at 16000 rpm for 10 min, wash twice with water, probe at 40 w for 3 s, and perform ultrasonic treatment 2 times to obtain the product). After treatment with the drugs for 2 h and 4 h respectively, discard the supernatant, wash twice with PBS, fix with paraformaldehyde for 15 min, stain with Hoechst for 5 min, wash three times with PBS and soak, and then place on a confocal microscope for photography (63× oil immersion lens).

[0080] The results are as Figure 5 shown. Fluorescently labeled siRNA indicates that the Pt-TPNs nanoparticles can interact with 143B, and the continuous penetration part proves that the nanoparticles enter the cells through the transcellular pathway. Moreover, the fluorescently labeled Pt-TPNs nanoparticles in 143B cells are more at 4 h than at 2 h, indicating that tumor cells have a strong uptake of Pt-TPNs.

[0081] Experimental Example 4

[0082] Investigate the escape of Pt-TPNs from the lysosomes of tumor cells

[0083] Seed 143B cells at 1×10 per well 5 in confocal dishes and allow them to adhere and grow for 24 h. Set up blank control, FAM-siRNA, and Pt-TPNs / FAM-siRNA groups. After treatment with the drugs for 0.5 h, 2 h, 4 h, and 8 h respectively, wash three times with PBS, add 500 μL of lysosome probe (dilute the probe to 60 μM using serum-free medium), incubate in the incubator for 50 min, wash three times with PBS, incubate with 300 μL of Hoechst for 25 min, wash three times with PBS and soak, and then place them under a confocal microscope for photography (63× oil immersion lens). The results are as follows Figure 6 shown. As the incubation time prolongs, gradually, the fluorescently labeled Pt-TPNs escape from the lysosomes into the cytoplasm, indicating that the Pt-TPNs nanoparticles have an obvious function of promoting lysosomal escape.

[0084] Experimental Example 5

[0085] Investigate the effect of Pt-TPNs on scavenging free radicals

[0086] Passage HK-2 cells, count the cells, and make the number of cells per well reach 1×10 5 . Seed them in 12-well plates, gently blow the culture medium with a tip to evenly distribute the cells, and then place them in the incubator for culture. After the cells adhere and grow for 24 h, discard the supernatant, and add 500 μL of DMEM / F12 complete medium, complete medium containing 12 μM cisplatin, and complete medium containing 12 μM Pt-TPNs respectively. After drug treatment for 24 h, discard the medium, wash three times with PBS, and add 3 kinds of free radical probes DCFH-DA, DHE, and DAF-DA respectively (dilute the concentrations of 10 μM DCFH-DA, 5 μM DHE, and 5 μM DAF-DA with phenol red-free DMEM / F12 containing 10% FBS), incubate in the incubator for 30 min, discard the supernatant, wash three times with PBS, add 500 μL of phenol red-free DMEM / F12 (10% FBS), and take pictures with a fluorescence microscope.

[0087] The results are as follows Figure 7 shown Figure 7 are the fluorescence images of Pt-TPNs scavenging intracellular ROS in HK-2 cells: blank control, cisplatin, and Pt-TPNs treatment groups. A, B, and C represent the levels of total intracellular ROS, ·O2 - , and ·NO respectively. Scale bar = 100 μm.

[0088] As can be seen from the figure, Pt-TPNs treatment can significantly eliminate excessive free radicals in cells and has good scavenging effects on total intracellular ROS, ·O2 - , ·NO.

[0089] Experimental Example 6

[0090] Investigate the inhibitory mechanism of Pt-TPNs on pyroptosis

[0091] 1) Determination of LDH, a marker of pyroptosis level in cells

[0092] Count 1×10 4 HK-2 cells per well were seeded in a 96-well plate and allowed to adhere for 24 h. A cell-free culture medium group (1% serum medium, the same for other wells), a drug-free blank control, a drug-free maximum lysis well, a blank control, a 45 μM cisplatin, and a 45 μM Pt-TPNs group were set up. After 48 h of drug treatment, 10 μL of release reagent was added to the maximum lysis well 1 h before detection, and the mixture was pipetted up and down and mixed well. The plate was then incubated in the incubator for another 1 h. The plate was then placed in a multi-well plate centrifuge at 400 g for 5 min. 90 μL of the supernatant was taken, and then 60 μL of the working solution was added, mixed well, incubated in the dark at room temperature for 30 min, and the absorbance was measured at 490 nm.

[0093] The results are as Figure 8 shown. Cisplatin caused the largest LDH release in HK-2 cells, indicating that cisplatin had the greatest toxicity to HK-2 cells. However, the LDH release of the Pt-TPNs of the present invention was significantly lower than that of cisplatin, indicating that Pt-TPNs significantly improved the toxicity of cisplatin to human renal tubular epithelial cells and reduced the level of pyroptosis.

[0094] 2) Study on the expression levels of proteins related to pyroptosis

[0095] (1) Cell administration and protein extraction:

[0096] HK-2 cells were digested and seeded in a six-well plate at a density of 10 6 per well and allowed to adhere and culture for 24 h. A blank control, a 45 μM cisplatin, and a 45 μM cisplatin + 100 μg / mL TPNs group were set up. After 48 h of drug treatment, protein extraction was performed as follows: The supernatant and PBS washing solution were collected, centrifuged at 4000 rpm for 5 min. The precipitate was dispersed with 150 μL of lysis buffer (RIPA:PMSF = 100:1). The lysis buffer was transferred to the corresponding well and lysed on ice for 5 min. Then, the cells were scraped with a 200 μL tip until the lysis buffer became viscous. The liquid in the well was transferred to a 1.5 mL centrifuge tube and lysed on ice for 30 min (vortexed every 10 min). Subsequently, the centrifuge tube was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected as the protein.

[0097] (2) Protein quantification:

[0098] Determined by the BCA protein quantification method: First, prepare 102 μL of working solution per well (100 μL of A + 2 μL of B (A:B = 50:1)) according to the BCA protein quantification kit instructions. Add 5 μL of the sample or 1 mg / mL standard protein to 100 μL of the working solution respectively, incubate in a water bath at 37 °C for 30 min. Then take out the sample and cool it to room temperature, and then measure the absorbance at 562 nm using a microplate reader (the lysis buffer is used as the blank). According to the absorbance, adjust the protein concentration of different groups to the same concentration with the lysis buffer.

[0099] (3) Protein treatment and preservation:

[0100] GSDME oligomerized protein sample: After adjusting the protein to the same concentration, add 2× non-reducing loading buffer according to the protein volume, vortex and mix well, and store at -20 °C.

[0101] Other protein samples: After adjusting the protein to the same concentration, add 5× reducing loading buffer (protein: loading buffer = 4:1), vortex and mix well, then boil it at 95 °C for 15 min, and store at -20 °C.

[0102] (4) Protein separation:

[0103] 1. Preparation: Preparation of 1 L of electrophoresis buffer - Dissolve 3.03 g of Tris, 14.4 g of Glycine, and 1 g of SDS in 1 L of water; Preparation of 1 L of transfer buffer - Dissolve 3.03 g of Tris and 14.4 g of Glycine in 200 mL of methanol and 800 mL of water.

[0104] 2. Gel preparation: Select an appropriate gel concentration according to the molecular weight. After preparation, quickly pour it into the sandwich plate, stop pouring the gel about 2 cm from the top of the short plate, add ultrapure water along the wall to press the gel for about 30 min. Wait until there is a clear boundary between the water and the gel, pour out the upper layer of water, and blot it dry with filter paper. Pour 5% separating gel to the top of the short plate, quickly insert the comb, and wait for 30 min until the upper layer of the gel solidifies.

[0105] 3. Loading and running the gel: After the gel is prepared, transfer the gel plate to the electrophoresis tank, add electrophoresis buffer, slowly pull out the comb, and gently blow the small holes with a 5 mL syringe to remove the air bubbles and residual gel in the small holes. Add 5 μL of marker to each of the two holes before and after the sample to monitor the protein molecule running process and protein separation. Load about 15 μg of the sample into the sample hole according to the protein concentration, and each hole does not exceed 40 μL. After loading, run the gel at 80 V for 30 min to align the proteins, and then adjust to 120 V and run the gel for about 1.5 h (the time is estimated according to the molecular weight of the target protein and the marker to separate proteins of different molecular weights as much as possible).

[0106] (5) Membrane transfer:

[0107] Before membrane transfer, activate the PVDF membrane with an appropriate amount of methanol for 1 min. According to the molecular weight of the target protein, discard the excess part of the gel. Place the gel and the membrane in the sandwich pattern of sponge - filter paper - gel - PVDF membrane - filter paper - sponge, then remove the air bubbles. Subsequently, put the membrane transfer clamp into the membrane transfer device and transfer the membrane at 4°C, 200 mA for 60 - 90 min.

[0108] (6) Blocking:

[0109] Wash the PVDF membrane three times with PBST, then soak the PVDF membrane in 5 mL of 5% skim milk powder (500 mg of milk powder dissolved in 10 mL of PBST), and incubate on a shaker at room temperature for 1 h.

[0110] (7) Antibody incubation and development:

[0111] Dilute the antibodies with 1% BSA (100 mg of BSA dissolved in 10 mL of PBST) (dilute GSDME and caspase - 3 at a ratio of 1:1000, and β - actin at a ratio of 1:5000), incubate overnight at 4°C, wash three times with PBST, then soak in 5 mL of secondary antibody diluted with PBST (rabbit anti - GSDME and caspase - 3; mouse anti - β - actin; both diluted at a ratio of 1:5000), and incubate on a shaker at room temperature for 1 h. Wash three times with PBST, add 200 μL of chemical developer (A:B = 1:1), and wait for 1 min for development.

[0112] It can be seen from Figure 9 that after treating HK - 2 cells with cisplatin, obvious cleavage of Caspase - 3 to produce Cleaved - Caspase - 3 occurs. Subsequently, Cleaved - Caspase - 3 cleaves the connection between GSDME - N and the C - terminus of GSDME, opening the auto - inhibitory structure of GSDME and releasing the N - terminus of GSDME. This terminus can bind to the phospholipids on the cell membrane, oligomerize to form pores, and extracellular water enters the cell, resulting in cell swelling and rupture. Intracellular LDH, immunogenic substances, etc. are released outside the cell, leading to an inflammatory environment. When intervened with TPNs, it does not affect the cleavage of Caspase - 3 and GSDME, and blocks pyroptosis by inhibiting the oligomerization of the GSDME - N terminus.

[0113] Experimental Example 7

[0114] Investigate the pharmacokinetic characteristics of Pt - TPNs and cisplatin

[0115] 1) Plasma concentration - time curve

[0116] Male rats weighing 180 - 200 g were purchased and raised in an adapted environment for one week. Two groups of cisplatin and Pt - TPNs were set up (n = 3). Tail vein injection was selected for drug administration, with a dose of 2.3 mg / kg Pt. Before tail vein injection, the rat tails were soaked in warm water at 45°C to soften the cutin layer, dilate blood vessels, select the bilateral veins, and use a No. 5.5 scalp needle for drug injection. The injection volume was about 1.2 mL. Orbital blood collection of 0.5 mL was performed at 0 h (before drug administration), 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 11 h, and 24 h into anticoagulant tubes, centrifuged at 3000 rpm for 10 min, about 200 μL of the supernatant was aspirated, 500 μL of aqua regia (concentrated hydrochloric acid: concentrated nitric acid = 3:1) was added, and nitrolysis was carried out in a fume hood for 48 h, centrifuged at 5000 rpm for 5 min, the supernatant was aspirated and added to 2.3 mL of water, centrifuged at 5000 rpm for 5 min, and the supernatant was aspirated for ICP determination.

[0117] 2) Tissue distribution

[0118] 143B cells in good culture status were placed in a culture dish. When the cells grew to 85% confluence, an appropriate amount of trypsin was added for digestion, centrifuged at 800 rpm for 5 min, washed once with PBS, and finally resuspended with an appropriate amount of PBS to prepare a cell suspension containing 5×10 6 143B cell suspension. 100 μL of the cell suspension was injected into the right abdomen of each mouse. A stable osteosarcoma model could be formed in about 2 weeks. When the tumor volume grew to about 200 mm 3 or so, the mice were randomly divided into 4 groups (cisplatin group at 6 h and 24 h, Pt - TPNs group at 6 h and 24 h), with four in each group. Tail vein injection was selected for drug administration, with a dose of 2.3 mg / kg Pt. The mice were euthanized at the end time, and tissues such as the heart, liver, spleen, lungs, kidneys, and tumors were collected and weighed. The tissues were placed in glass tubes respectively, 3 mL of aqua regia was added, and they were incubated in a water bath at 60°C for 24 h. 500 μL of the digestion solution was aspirated and added to 2.5 mL of purified water for ICP determination.

[0119] The results of the blood drug concentration - time curve are as Figure 10 shown in a. As the time after drug administration increased, the concentration of cisplatin in the rat blood decreased rapidly, from 5 μg / ml to approaching 0 within 3 h, while Pt - TPNs could still be detected in the blood within 25 h, indicating good drug metabolism of Pt - TPNs; the tissue distribution is as Figure 10 shown in b and 11c. After 6 h of drug administration, the content of cisplatin in the kidneys was the highest, and as the drug administration time extended, after 24 h of drug administration, the content of cisplatin in the kidneys did not decrease and remained at about 2 μg / g; the content of Pt - TPNs of the present invention in the kidneys was much lower than that of cisplatin after 6 h and 24 h of drug administration, indicating that Pt - TPNs can reduce the renal injury of cisplatin.

[0120] Experimental Example 8

[0121] Investigation on the alleviation of renal toxicity of Pt-TPNs in vivo:

[0122] 1) Determination of renal injury factors: After collecting plasma, the determination was carried out according to the Cre and BUN detection kits.

[0123] 2) Determination of kidney index: After the mice were euthanized, the body weight was weighed, the kidneys were collected and weighed, and the kidney index was calculated using the ratio of kidney weight to the corresponding mouse body weight.

[0124] 3) HE staining of the kidney: After the mice were euthanized, the kidneys were collected, fixed with paraformaldehyde for 24 h, embedded in paraffin, sectioned, stained with HE, and scanned and photographed.

[0125] 4) Renal injury indicators (immunohistochemistry of Kim and NGAL): After the mice were euthanized, the kidneys were collected, fixed with paraformaldehyde for 24 h, sectioned, incubated with Kim and NGAL antibodies respectively, and scanned and photographed.

[0126] The results are as Figure 11 shown. Cisplatin significantly increased the levels of renal injury factors Cre and BUN in vivo, and the kidney index was as high as 0.018. However, Pt-TPNs of the present invention significantly reduced the levels of Cre and BUN and the kidney index in vivo, indicating that Pt-TPNs significantly reduced the renal injury caused by cisplatin.

[0127] HE staining showed that Pt-TPNs significantly reduced the renal vacuolar degeneration caused by cisplatin and reduced the pathological manifestations.

[0128] The IHC results showed that Pt-TPNs significantly reduced the high expression of Kim and NGAL caused by cisplatin in renal tissues, and the renal injury index decreased from 8 to 3.

[0129] Experimental Example 9

[0130] Investigation on the effect of Pt-TPNs in alleviating oxidative stress in vivo

[0131] 1) Determination of renal oxidative level: Accurately weigh the weight of renal tissue, add the corresponding PBS solution (protease inhibitor was added to PBS in advance) according to the ratio of weight (g): volume (mL) = 1:9, homogenize, centrifuge at 2500 rpm for 10 min, collect the supernatant, and carry out the determination according to the GSH and CAT detection kits.

[0132] 2) Determination of renal lipid oxidation products: After collecting plasma, the determination was carried out according to the MDA detection kit.

[0133] 3) Determination of renal nucleic acid and protein oxidation products: After euthanasia of mice, kidneys were collected, fixed with paraformaldehyde for 24 h, sliced, incubated with Nitro tyrosine and 8-OHdG antibodies, respectively, and scanned and photographed.

[0134] The results are as follows Figure 12 As shown, cisplatin caused an increase in the content of GSH and CAT in the kidney, indicating that the free radicals in the renal tissue could not be processed in time, triggering lipid peroxidation, resulting in an increase in the MDA content in the renal tissue of rats, and the Pt-TPNs of the present invention significantly reversed this oxidative stress damage.

[0135] Experimental Example 10

[0136] Investigate the effect of Pt-TPNs in clearing inflammatory factors in vivo

[0137] The tissue was rinsed with pre-cooled PBS to remove residual blood, and the tissue was cut into pieces after weighing. The tissue was added into a glass homogenizer at a ratio of tissue weight to PBS volume of 1:8, and the tissue was ground and lysed on ice. Ultrasound was then used for 60w, 9s, 3 times, and the homogenate was centrifuged at 5000g for 10min. The supernatant was taken and diluted with the universal diluent provided in the kit (diluted 1500 times), and the IL-1α, IL-1β, TNF-α and IL-6 ELISA detection kits were used for determination.

[0138] The results are as follows Figure 13 As shown, cisplatin significantly increased the levels of inflammatory-related factors IL-1α, IL-1β, TNF-α and IL-6 in kidney tissue, while the Pt-TPNs of the present invention significantly reduced the inflammatory damage caused by cisplatin.

[0139] Experimental Example 11

[0140] Investigating the effect of Pt-TPNs on inhibiting cell pyroptosis in vivo

[0141] After collecting plasma, the LDH test kit was used to measure

[0142] The results are as follows Figure 14 As shown, cisplatin significantly increased the LDH content in plasma, which indicated the occurrence of cell pyroptosis, while the Pt-TPNs of the present invention significantly inhibited the cell pyroptosis caused by cisplatin.

[0143] Experimental Example 12

[0144] Study on the anti-tumor effect of Pt-TPNs

[0145] The length and width of the tumor were measured daily using a vernier caliper, and the tumor volume was calculated using a formula.

[0146] The results are as follows Figure 15As shown, cisplatin can significantly reduce tumor growth and has excellent anti-tumor effects. There is no significant difference in the anti-tumor effects between Pt-TPNs and cisplatin.

[0147] Experimental Example 13

[0148] Investigate the safety of Pt-TPNs

[0149] Weigh the mice every day from the start of administration and record.

[0150] The results are as Figure 16 shown. Cisplatin reduced the body weight of the mice, indicating that cisplatin has an adverse effect on the body mechanism of the mice and affects biological safety. However, there is no significant difference in the body weight between the mice administered with Pt-TPNs of the present invention and normal mice, indicating that Pt-TPNs have high biological safety.

[0151] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity, characterized in that It includes epigallocatechin gallate and cisplatin. The nanoparticles are formed by the self-polymerization of epigallocatechin gallate and cisplatin through catalytic oxidation by Mn under a weak alkaline environment after bonding. 2+ ​ The cisplatin is carboxylated cisplatin, and the carboxylated cisplatin is bonded to the epigallocatechin gallate through a carboxyl group.

2. The nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity according to claim 1, characterized in that The Mn 2+ catalyst is MnCl 2· 4H2O, and the weak alkalinity is pH 7.4 - 8.

5.

3. A method for preparing a nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity according to any one of claims 1-2, characterized in that It includes the following steps: S1. Oxidize cisplatin with H2O2, cool after vigorous stirring, wash and then freeze-dry to obtain the primary product Pt(OH)2; S2. Dissolve Pt(OH)2 and succinic anhydride in DMSO, vigorously stir and then freeze-dry, and then inject acetone to precipitate crystals at low temperature. Dry the crystals to obtain carboxylated cisplatin Pt(COOH)2; S3. Mix Pt(COOH)2, EDC · HCl, and DMAP, add DMSO and stir in a water bath, then add EGCG and continue stirring to obtain tea polyphenol Pt-EGCG bonded with cisplatin; S4. Add the Pt-EGCG reaction solution to a weakly alkaline buffer solution, add Mn and PVP under stirring, centrifuge after water bath, and perform ultrasonic treatment to obtain the nanoparticles Pt-TPNs that multi-dimensionally reduce the nephrotoxicity of cisplatin. 2+ ​ 4. The preparation method according to claim 3, characterized in that In the step S1, the concentration of H2O2 is 30%, and the stirring temperature is 45 °C.

5. The preparation method according to claim 3, characterized in that In the step S2, the molar ratio of Pt(OH)2 to succinic anhydride is 1:3; the stirring temperature is 70 °C.

6. The preparation method according to claim 3, characterized in that In the step S3, the molar ratio of Pt(COOH)2 to EGCG is 1:1 to 2; the water bath temperature is 27 °C.

7. The preparation method according to claim 3, characterized in that In the step S4, the molar ratio of Pt-EGCG to Mn 2+ is 10:1 to 1:

10.

8. Use of a nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity according to any one of claims 1-2 or a nanoparticle for multi-dimensionally reducing cisplatin nephrotoxicity prepared by the preparation method according to any one of claims 3-7 in the preparation of an anti-tumor drug.

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