Preparation of phenol chemically modified cis-platinum material and nucleic acid-loaded composite material thereof, and application of phenol chemically modified cis-platinum material in tumor resistance

Abstract

The invention belongs to the field of medicines, and particularly discloses a pharmacodynamic active phenol chemical modified cis-platinum material which is at least one compound with a structural general formula 1, namely imgabs0 #. The invention also discloses preparation and application of the material. The method provided by the invention can effectively reduce toxicity and can synergistically improve the drug effect performance.

Description

Technical Field

[0001] The present invention belongs to the field of drugs, and particularly relates to the field of anti-tumor drugs. Background Art

[0002] At present, cisplatin is one of the first-line anti-cancer drugs for the clinical treatment of various malignant tumors, but there are problems of multi-drug resistance, which seriously affect the clinical efficacy and long-term survival rate of patients. At the same time, there are various toxic and side effects in cisplatin chemotherapy, such as nephrotoxicity, neurotoxicity, etc., which severely limit its clinical application. Therefore, it is very important to improve the sensitivity of cisplatin to tumors and reduce its toxic and side effects at present. Gene therapy is still in the research and experimental stage in clinical application. At present, only some gene therapy programs have been approved for the treatment of specific types of tumors. However, as a highly efficient and precise emerging technology, gene therapy has a broader development prospect.

[0003] Gene therapy is a technology that uses therapeutic nucleic acids to intervene, including small interfering RNA (siRNA), antisense oligonucleotides, miRNA, etc. These therapeutic nucleic acids can selectively inhibit or activate specific genes, thereby treating cancer. They can interfere with the signal transduction pathway in cells and affect the growth and differentiation of cancer cells by targeting specific targets. For example, antisense oligonucleotides can target different cancer-related genes in gene therapy, inhibit the proliferation and growth of tumor cells, and thus have a certain anti-cancer effect.

[0004] In terms of the modification methods of cisplatin drugs, current related research includes but is not limited to: optimizing the targeted delivery system of cisplatin to improve the efficacy of cisplatin and reduce its side effects; searching for compounds that can reduce cisplatin resistance to improve the therapeutic effect of cisplatin; searching for combination drug strategies to improve the efficacy and reduce side effects.

[0005] For example, the US patent document with the publication number US20210393785A1 discloses a nucleic acid nanocarrier drug and its preparation method, which records that a pharmaceutically active molecule is modified on a nucleic acid nanoparticle, and the pharmaceutically active molecule includes cisplatin. Another example is that the Chinese patent document with the publication number CN 111150852 A reports a drug including a nucleic acid nanoparticle and cisplatin, and cisplatin is mounted on the nucleic acid nanoparticle.

[0006] In summary, although the prior art has reported means of sensitizing cisplatin through nucleic acids and has also reported that other active ingredients can be further introduced on this basis, in the prior art, the combination of components is mainly based on hydrogen bonds or compatibility methods, and the drug loading, targeting, and drug efficacy need to be improved. Moreover, the metabolic behaviors of the components are not uniform, which results in unsatisfactory drug metabolism and toxic and side effects. Summary of the Invention

[0007] In view of the problems existing in the prior art, the first object of the present invention is to provide a pharmaceutically active phenol chemically modified cisplatin material, aiming to provide a composite material with good co-metabolism behavior and excellent drug-loading prospects.

[0008] The second object of the present invention is to provide the preparation and application of the pharmaceutically active phenol chemically modified cisplatin material.

[0009] The third object of the present invention is to provide a composite material containing nucleic acid supported by the pharmaceutically active phenol chemically modified cisplatin material and its preparation application in anti-tumor drugs.

[0010] A pharmaceutically active phenol chemically modified cisplatin material is at least one compound having the general formula of Structure 1;

[0011]

[0012] In the said Structure 1, R1 is alkylene, substituted alkylene, alkenylene, arylene or cycloalkylene;

[0013] R2 is the esterification residue of the pharmaceutically active phenol (referring to the remaining group fragment after deducting the OH for esterification in the phenol substance);

[0014] R3 is H or a substituent of Formula A;

[0015]

[0016] In view of the problems such as the inconsistent metabolism behaviors of the pharmaceutically active phenol and cisplatin and the lack of obvious effect on reducing the toxicity and enhancing the efficacy of cisplatin, the present invention provides a brand-new pharmaceutically active phenol chemically modified cisplatin material, which includes hydroxylated cisplatin (cisplatin prodrug), a carboxyl-containing linking fragment based on ester group chemical modification, and a pharmaceutically active phenol (abbreviated as phenol or phenol substances) esterified with the carboxyl group of the linking fragment. The material of the present invention innovatively chemically modifies the pharmaceutically active phenol on the cisplatin prodrug, so as to improve the metabolic unity of cisplatin and phenol and the cisplatin toxicity reduction effect. In addition, it can self-assemble into a nanocage with a large loading chamber and abundant active groups, which is beneficial to improving the drug-loading amount based on physical and chemical methods, and is also beneficial to reducing the risk of the loaded components becoming ineffective, and thus is beneficial to synergistically improving the drug efficacy.

[0017] In the present invention, the combination of the structures and fragments of the compounds of Structure 1 is the key to synergistically improving their performance.

[0018] In the present invention, R1 is alkylene with 1 to 6 carbon atoms or substituted alkylene with substituents on its carbon chain;

[0019] The substituent is at least one of alkyl, alkoxy, halogen, phenyl, nitro, and alkenyl;

[0020] Preferably, R1 is an alkylene group having 2 to 4 carbon atoms.

[0021] In the present invention, the pharmaceutically active phenol is a phenolic substance having at least one of anti-inflammatory, anti-tumor, and antioxidant activities, preferably at least one of tea polyphenols, catechins, catechin-3-gallol, catechin-3-gallate, gallic acid, caffeic acid, flavanones, flavonoids, and anthocyanins.

[0022] The present invention also provides a method for preparing the pharmaceutically active phenol chemically modified cisplatin material, which comprises subjecting cisplatin to hydroxylation treatment, followed by esterification carboxylation treatment with a carboxylated raw material, and then esterification treatment with a pharmaceutically active phenol.

[0023] The carboxylated raw material is at least one compound having the structure of Formula 2 or Formula 3;

[0024]

[0025] In Formula 2, R4 and R5 are each independently at least one of a halogen and a hydroxyl group.

[0026] In the present invention, cisplatin is innovatively subjected to hydroxylation, carboxylation, and esterification modifications, so as to achieve the chemical modification of cisplatin prodrug and pharmaceutically active phenol, thereby facilitating the improvement of its properties.

[0027] In the present invention, cisplatin and an oxidant are mixed for hydroxylation treatment;

[0028] In the present invention, the oxidant is hydrogen peroxide;

[0029] In the present invention, the conditions for the hydroxylation reaction are 30 to 60 °C;

[0030] In the present invention, the molar ratio of the product of the hydroxylation treatment to the carboxylated raw material is 1:2 to 3;

[0031] In the present invention, there is no special requirement for the temperature in the esterification carboxylation treatment stage. For example, it can be 10 to 40 °C, and further can be room temperature;

[0032] In the present invention, the molar ratio of the product of the esterification carboxylation to the pharmaceutically active phenol is 1:2 to 3.

[0033] In the present invention, when R4 and R5 in Formula 2 are H, during the esterification stage, activation treatment can be carried out according to the conventional esterification requirements.

[0034] The present invention also provides an application of the pharmaceutically active phenol chemically modified cisplatin material, which is used as a carrier for compounding pharmaceutically active auxiliary components to prepare an anti-tumor drug;

[0035] Preferably, the pharmacodynamic adjuvant components include at least one of anti-tumor active components, drug sensitivity components, and targeting aids;

[0036] More preferably, the drug sensitivity component includes drug sensitivity nucleic acid.

[0037] In the present invention, the nucleic acid may be a nucleic acid known in the industry that can enhance sensitivity, and it can be conventionally selected and replaced according to different cancers. For example, the nucleic acid includes at least one of, but is not limited to, antisense oligonucleotides, small interfering RNAs, messenger RNAs, plasmids, etc.

[0038] The present invention also provides a nucleic acid-active phenolic chemically modified cisplatin composite anti-tumor active component, a nano-cage assembled by the pharmacodynamic active phenolic chemically modified cisplatin material, and a nucleic acid filled therein.

[0039] The compound of formula 1 according to the present invention can self-assemble to construct a large-chamber nano-cage, and rich active groups are distributed on the skeleton of the nano-cage. Thus, the filling and loading of nucleic acids can be improved based on physical and chemical bond methods. Moreover, it is also beneficial to reduce the degradation and failure problem of nucleic acids during the treatment stage, thereby synergistically improving the drug effect. Moreover, it is also beneficial to improve the loading capacity.

[0040] In the present invention, the nucleic acid is combined with the active phenolic chemically modified cisplatin through the action of van der Waals forces, hydrogen bonds, and chemical bonding;

[0041] Preferably, in the nucleic acid-active phenolic chemically modified cisplatin composite anti-tumor active component, the encapsulation efficiency is greater than 70%.

[0042] For example, in a typical material of the present invention, after the nucleic acid is encapsulated in cisplatin-bonded tea polyphenol self-assembled nanoparticles (pharmacodynamic active phenolic chemically modified cisplatin material), it can effectively avoid being degraded by nucleases in the in vivo environment; it can ensure that Pt-TPNs can successfully pass through the blood circulation system to target organ tissues and be successfully taken up by cells after embedding the corresponding nucleic acid molecules; the nanoparticles can successfully release cisplatin and nucleic acids after being taken up by cells and play a biological function; the specific biological role of the nucleic acid molecule targeting the drug-resistant gene in the nanomaterial is to increase the drug sensitivity of cisplatin.

[0043] In the present invention, other well-known anti-tumor active components are also allowed to be included. For example, docetaxel.

[0044] In the present invention, the targeting aid may be a component known in the industry that is beneficial to improving the drug targeting ability. For example, it may be drugs targeting the PI3K / Akt / mTOR signaling pathway such as PPT-086 and AS703026.

[0045] In the present invention, the anti-cancer means described above can also be used in combination with existing anti-cancer means, such as immunotherapy.

[0046] The present invention also provides a preparation method of the nucleic acid-active phenolic chemically modified cisplatin composite anti-tumor active ingredient. The method comprises self-assembling the pharmaceutically active phenolic chemically modified cisplatin material and the nucleic acid to obtain the composite.

[0047] The present invention also provides an application of the nucleic acid-active phenolic chemically modified cisplatin composite anti-tumor active ingredient in the preparation of anti-tumor drugs.

[0048] The present invention also provides an anti-tumor drug, which contains a pharmaceutically effective amount of the nucleic acid-active phenolic chemically modified cisplatin composite anti-tumor active ingredient;

[0049] The drug of the present invention further contains pharmaceutically acceptable excipients;

[0050] The drug of the present invention has a pharmaceutically acceptable dosage form, such as an injection dosage form, an oral preparation, a topical preparation, an inhalant, a mucosal administration preparation, etc.

[0051] Beneficial effects

[0052] The present invention provides a chemically modified pharmaceutically active phenolic chemically modified cisplatin material, which has good metabolic synchrony, helps to improve the cisplatin attenuation effect. In addition, it is also beneficial to construct nanocages with large chambers and high chemical activity, so as to facilitate the physical and chemical loading of active ingredients such as nucleic acids, and help to improve the drug loading effect and drug efficacy.

[0053] In the present invention, the chemically modified pharmaceutically active phenolic chemically modified cisplatin material and the nucleic acid are innovatively compounded, and they can be compounded based on hydrogen bonds and chemical bonds other than van der Waals forces, which helps to improve the drug loading capacity, improve the activity, and in addition, is also beneficial to reducing the toxic and side effects. Description of the drawings

[0054] Figure 1 It is a synthesis schematic diagram of Pt-TPNs / siRNA nanoparticles;

[0055] Figure 2 It is the particle size (A), zeta potential (B), siRNA loading capacity (C, D), structure under scanning electron microscopy and elemental analysis results (E) of Pt-TPNs / siRNA nanoparticles;

[0056] Figure 3 It is the stability (A) of Pt-TPNs / siRNA nanoparticles in different media and the ability (B) to release Pt and siRNA under the simulated intracellular environment at the test tube level

[0057] Figure 4 Cell uptake and lysosomal escape experiments showed the effective release diagram of siRNA;

[0058] Figure 5 RT-PCR (A) and WB (B) experiments were used to verify its ability to knockdown the target gene. MTT (C, D) and flow cytometry experiments were used to verify the sensitization effect of Pt-TPNs / siRNA (E).

[0059] Figure 6 Tissue distribution was used to understand the in vivo delivery distribution and enrichment of Pt-TPNs / siRNA; the distribution and enrichment of cisplatin and Pt-TPNs / siRNA in various organs in vivo over time (A, B);

[0060] Figure 7 In a tumor-bearing mouse model, the sensitization effect of Pt-TPNs / siRNA (A, B), TUNEL immunofluorescence staining proved its sensitization effect (C), and immunohistochemistry verified the effectiveness of its knockdown protein (D);

[0061] Figure 8 Blood biocompatibility hemolysis experiment (A), effect on body weight (B), and effect on the main metabolic organ kidney (C, D) of the nanoparticles

[0062] Figure 9 Diagram of the role of cisplatin and cisplatin + TPNs physical mixture in solving renal toxicity oxidative stress (A). Detailed implementation manners

[0063] siRNA is used as the preferred nucleic acid molecule to be loaded because of its high-efficiency knockout effect on the target gene, relatively small particle size for easy encapsulation, relatively good biological safety, low cost, etc.; as a typical example, the inventive concept of the present invention is directed to the sensitizing nucleic acid molecule siRNA targeting the osteosarcoma cisplatin chemotherapy resistance gene CSNK2A1. In the following cases, unless otherwise stated, it all refers to siCSNK2A1 (siRNACSNK2A1);

[0064] The above cases are only used to illustrate the present invention, but not to 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 shall fall within the scope of the present invention. Detailed implementation manners

[0066] 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.

[0067] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or substitution made to the method, steps or conditions of the present invention shall fall within the scope of the present invention.

[0068] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art; unless otherwise specified, the reagents used in the examples are all commercially available.

[0069] The following are the specific implementation methods:

[0070] Example 1

[0071] Preparation of Pt-TPNs / siRNA:

[0072] (1) Synthesis of carboxylated cisplatin

[0073] Precisely weigh 500 mg of cisplatin into a round-bottom flask, add 20 mL of H2O2 (30%), seal it with a sealing film, pierce 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. Precisely weigh 333 mg of hydroxylated cisplatin (1 mmol) and 302.1 mg of succinic anhydride (3 mmol), 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, and dry the crystals in a vacuum drying oven at 50 °C for 3 h, weigh, and approximately obtain 510 mg of carboxylated cisplatin.

[0074] (2) Preparation of Pt-TPNs

[0075] Weigh 53.4 mg of carboxylated cisplatin, 38.28 mg of EDC·HCl, and 18.32 mg of DMAP (4-dimethylaminopyridine) into a 10 mL EP tube, add 3 mL of DMSO, stir in a 27 °C water bath for 3 h, add 137.52 mg of EGCG (tea polyphenols), and stir for 24 h to obtain the Pt-EGCG reaction solution.

[0076] Then add 60 μL of the Pt-EGCG (also known as PtCOOH-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, keep in a 27 °C water bath for 3 h, centrifuge at 16000 rpm for 10 min, wash twice with water, and ultrasonicate twice with a 40 w probe for 3 s to obtain Pt-TPNs nanoparticles (also known as Pt-TPNs).

[0077] Preparation of TPNs (EGCG self-assembled nanoparticles): 2.4 mL of HEPES (pH 8.0, 10 mM) buffer and 60 μL of EGCG were added with 48 μL of MnCl2·4H2O and 20 μL of 20% PVP under magnetic stirring. The mixture was incubated in a water bath at 27 °C for 3 h, centrifuged at 16,000 rpm for 10 min, washed twice with water, and sonicated twice with a probe at 40 W for 3 s to obtain TPNs nanoparticles.

[0078] (3) Determination of drug loading

[0079] Formulation for the preparation of Pt-TPNs / siRNA:

[0080] According to the results of the co-loading experiment of siRNA, the final formulation was confirmed as follows: In a 2 mL EP tube, 240 μL of HEPES (8.0, 10 mM) buffer, 6 μL of Pt-EGCG reaction solution, and 9.6 μL of 100 μM siRNA were added. The mixture was cross-linked by magnetic stirring for 30 min, then 4.8 μL of MnCl2·4H2O and 20 μL of 20% PVP were added. The mixture was incubated in a water bath at 27 °C for 2.5 h, centrifuged at 16,000 rpm for 10 min, washed twice with water, and sonicated twice with a probe at 40 W for 3 s to obtain the product.

[0081] Determination of the carried Pt(Ⅳ): 4.8 mL of Pt-TPNs were prepared and freeze-dried to weigh the nanoparticles. 1 mL of the nanoparticles was centrifuged at 16,000 rpm for 10 min to remove the supernatant, 200 μL of 30% nitric acid was added, and the mixture was placed in a fume hood with the lid open for digestion for 48 h. The digested solution was added to 3 mL of water for ICP determination. The measurement result was A. The formula for calculating the Pt loading was: 3.2×A / weight of nanoparticles per milliliter. (Configuration of the ICP standard curve: 10 mg of cisplatin was accurately weighed and added to 200 μL of concentrated nitric acid. The mixture was boiled in a water bath at 70 °C until clear, and water was added to 1 mL to prepare a 10 mg / mL mother liquor. The standard curve concentrations were configured by the two-fold dilution method to be 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.625 μg / mL, 0.78125 μg / mL, 0 μg / mL).

[0082] Determination of the siRNA loading capacity: The encapsulation of siCSNK2A1 in Pt-TPNs / siRNA was examined by gel electrophoresis experiment. Figure 5It can be seen that when the input amounts of siRNA are 0.2 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM respectively, the encapsulation efficiency is greater than 70%, and when the input amount is 4 μM, the drug loading amount is the highest, which is 3.560 μM. Therefore, the input amount of siCSNK2A1 is selected as 4 μM for the preparation of the final formulation of Pt-TPNs / siRNA. Mass spectrometry and hydrogen spectrum are used for the characterization of Pt(Ⅳ); particle size, zeta potential, submicroscopic structure under transmission electron microscopy, elemental analysis, etc. are used to distinguish the empty carrier and Pt-TPNs / siRNA nanoparticles.

[0083] (3) Stability determination

[0084] Detect the change of the volume of nanoparticles with time in water, hepes buffer, complete DMEM medium, and normal saline (saline).

[0085] (4) Drug release ability determination

[0086] It is divided into an extracellular environment group with pH 7.4 and a simulated intracellular environment group with pH 7.4 + GSH. The release of Pt and siRNA from the nanoparticles in the two environments is measured respectively.

[0087] Figure 1 Schematic diagram of the synthesis mode of Pt-TPNs / siRNA nanoparticles

[0088] Figure 2 For Pt-TPNs / siRNA nanoparticles, particle size (A): The particle size of the nanoparticles after loading siRNA is 200 nm. Zeta potential (B): The zeta potential after loading siRNA is -20 mV. siRNA loading capacity (C, D): After the input of siRNA at different concentrations, the encapsulation efficiency of the nanoparticles is greater than 70%, and when the input is 4 μM, the drug loading amount is the highest; and elemental analysis (E): Elements such as C, N, O, Pt, and P are evenly distributed in the nanoparticles. The above results show the successful loading of Pt and siRNA, and the nanoparticles meet the standards.

[0089] Figure 3 For the stability of Pt-TPNs / siRNA nanoparticles in different media (A): The nanoparticles can stably exist in different media; the ability to release Pt and siRNA under the simulated intracellular environment at the test tube level (B): After adding glutathione, which simulates the intracellular environment, it has obvious release performance. It shows that the nanoparticles can be effectively released in the in vivo cell environment.

[0090] Example 2

[0091] Investigate the in vitro nucleic acid delivery effect of Pt-TPNs / siRNA in Example 1:

[0092] (1) Cellular uptake: Seed 143B cells at a density of 1×10⁵ cells per well in a confocal dish and allow them to adhere and grow for 24 h. Set up blank group, FAM-siRNA (i.e., the above-mentioned siRNACSNK2A1), and Pt-TPNs / FAM-siRNA (i.e., the above-mentioned Pt-TPNs / siRNA) groups. After treatment with the respective drugs for 2 h and 4 h, 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 take pictures under a confocal microscope (63× oil immersion objective). Secondly, seed 143B cells at a density of 1×10⁵ cells per well in a 6-well plate and allow them to adhere and grow for 24 h. Set up blank group, FAM-siRNA, and Pt-TPNs / FAM-siRNA groups. After treatment with the respective drugs for 2 h and 4 h, discard the supernatant, wash twice with PBS, digest the cells with 200 μL of trypsin, add 1 mL of complete medium to terminate digestion, collect the liquid, centrifuge at 1000 rpm for 5 min, wash twice with PBS, resuspend with 400 μL of PBS, and detect the fluorescence intensity by flow cytometry.

[0093] (2) Lysosomal escape: Seed 143B cells at a density of 1×10⁵ cells per well in a confocal dish and allow them to adhere and grow for 24 h. Set up blank group, FAM-siRNA, and Pt-TPNs / FAM-siRNA groups. After treatment with the respective drugs for 0.5 h, 2 h, and 8 h, wash three times with PBS, add 500 μL of lysosomal probe (dilute the probe to 60 μM with serum-free medium), incubate in an 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 take pictures under a confocal microscope (63× oil immersion objective).

[0094] Figure 4 Cellular uptake and lysosomal escape experiments show the effective release of siRNA; Cellular uptake (A): (a) Laser confocal microscopy images (blue: nucleus; green: Pt-TPNs / FAM-siRNA; scale bar = 20 μm), (b) flow cytometry plots, and (c) fluorescence quantification plots of 143B cells taking up Pt-TPNs / siRNA, Lysosomal experiment (B): The behavioral trajectory of Pt-TPNs / siRNA in 143B cells (blue: nucleus; green: Pt-TPNs / FAM-siRNA; red: lysosome; scale bar: 20 μm). The experimental results show that the nanoparticles can be effectively taken up by cells and released from lysosomes into the cells to take effect.

[0095] Example 3

[0096] Investigate the vector performance of Pt-TPNs / siRNA in Example 1:

[0097] (1) Ability to knockdown target genes

[0098] Using the osteosarcoma 143B cell line as an example cell, seed 1.5×10 6 cells per well into a 6-well plate, treat with Pt-TPNs / siRNA (CPt = 12 μM) for 48 hours, lyse with 50 μL of RIPA lysis buffer on ice for 30 minutes, sonicate at 60 W for 3 seconds, centrifuge at 12,000 rpm for 10 minutes, take the supernatant, add SDS, and boil and denature at 100 °C for 10 minutes. Verify the effect of knocking down the target gene by WB electrophoresis experiment.

[0099] Using the osteosarcoma 143B cell line as an example cell, seed 1.5×10 6 cells per well into a 6-well plate, treat with Pt-TPNs / siRNA (CPt = 12 μM) for 48 hours, then extract RNA. After reverse transcription, verify the effect of knocking down the target gene by RT-PCR experiment.

[0100] (2) Sensitization effect after knocking out sensitizing genes

[0101] Using the osteosarcoma 143B cell line as an example cell, seed 143B cells (5×10 3 cells per well) onto a 96-well plate, let them adhere for 24 h, set up the TPNs group (blank group, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL), cisplatin group (blank group, 2 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 45 μM, calculated by Pt content), Pt-TPNs group (concentration set the same as cisplatin), Pt-TPNs / siRNA group (concentration set the same as cisplatin). After drug treatment for 48 h, discard the supernatant, wash twice with PBS, add 100 μL of 0.5 mg / mL MTT, incubate in an incubator for 4 h, discard the supernatant, add 150 μL of DMSO, shake on a shaker at 37 °C for 20 min, and measure the absorbance at 490 nm.

[0102] For flow cytometry apoptosis detection, seed 1×105 143B cells per well into a 12-well plate, let them adhere for 24 h, and set up the blank group (without dye), single stain FITC, single stain PI, blank group, cisplatin (CPt = 30 μM), Pt-TPNs (CPt = 30 μM), and Pt-TPNs / siRNA (CPt = 30 μM) groups respectively. After drug treatment for 48 h, discard the supernatant, wash twice with PBS, digest with trypsin without EDTA, terminate digestion with 1 mL of PBS, centrifuge at 1000 rpm for 5 min, wash twice with PBS, stain according to the apoptosis kit, and immediately perform flow cytometry (store at 4 °C).

[0103] Figure 5The ability of knocking down the target gene was verified by RT-PCR (A) and WB (B) experiments: The experiment showed that the loaded siRNA effectively knocked down the mRNA expression and protein expression of the target gene. The sensitization effect of Pt-TPNs / siRNA was verified by MTT (C, D) and flow cytometry experiments (E): The experimental results showed that since the siRNA target gene was a sensitizing gene for cisplatin, based on the above experimental results, the nanoparticles could significantly promote the sensitivity of osteosarcoma cells to cisplatin.

[0104] Example 4

[0105] Investigate the in vivo nucleic acid delivery effect of Pt-TPNs / siRNA:

[0106] (1) Tissue distribution

[0107] The in vivo nucleic acid delivery effect was verified using a subcutaneous model of osteosarcoma mice. After injecting the osteosarcoma model via the tail vein, at 6 hours and 24 hours later, the heart, liver, spleen, lungs, kidneys, and tumors were taken. Each organ was digested with 3 ml of aqua regia, and then 500 μl was taken and added to 2500 μl of double-distilled water. The content of Pt in the organ was measured using ICP (inductively coupled plasma atomic emission spectrometry) to understand its in vivo delivery distribution and metabolism.

[0108] Figure 6 Tissue distribution to understand the in vivo delivery distribution and metabolism of Pt-TPNs / siRNA: The experiment showed that compared with cisplatin, the nanoparticles could be highly enriched at the tumor site, and the half-life of the nanoparticles at the tumor site was long, with a longer-lasting drug effect.

[0109] Example 5

[0110] Investigate the intrinsic biological activity of in vivo tumor sensitization of Pt-TPNs / siRNA:

[0111] (1) Tumor-bearing mouse model

[0112] The well-cultured 143B cells were placed in a culture dish. When the cells reached 75% confluence, an appropriate amount of trypsin was added for digestion. After centrifugation at 800 rpm for 5 min, they were washed once with PBS, and finally resuspended with an appropriate amount of PBS to prepare a cell suspension containing 5×10 6 143B cells in 100 μL. 100 μL of the cell suspension was injected into the right abdomen of each mouse, and a stable osteosarcoma model could be formed in about 2 weeks. When the tumor volume grew to 100 mm 3Mice were randomly divided into four groups (n = 5), namely the normal saline group, the cisplatin group (CPt = 2 mg / kg), the Pt-TPNs group (CPt = 2 mg / kg), and the Pt-TPNs / siRNA group (CPt = 2 mg / kg). Drugs were injected via the tail vein on days 0, 2, and 4, and the experiment ended on day 16.

[0113] (1) Immunofluorescence and immunohistochemistry

[0114] TUNEL immunofluorescence staining and immunohistochemistry of the target gene were used to explore the sensitization effect and the effect of knocking down the target gene.

[0115] Figure 7 In the tumor-bearing mouse model, the sensitization effect of Pt-TPNs / siRNA (A, B): This experiment showed that compared with the cisplatin group, the nanoparticle group more effectively inhibited tumor growth. TUNEL immunofluorescence staining proved its sensitization effect (C): This experiment showed that after loading the sensitizing siRNA, the sensitivity of osteosarcoma to cisplatin could be promoted. Immunohistochemistry verified the effectiveness of knocking down the protein (D): This experiment showed that after loading the sensitizing siRNA, the siRNA could play a role and effectively knock out the target gene.

[0116] Example 6

[0117] Investigate the in vitro and in vivo biosafety of Pt-TPNs / siRNA:

[0118] (1) Hemolysis experiment

[0119] One healthy C576J black mouse was selected, and about 500 μL of blood was taken from the eyeball and placed into an anticoagulation tube. It was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and it was repeatedly rinsed 4 times with normal saline and then resuspended with 25 mL of normal saline to prepare a 2% blood cell concentration. Normal saline, Water, Pt-TPNs / siRNA (800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL), and the no red blood cell background group (800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL) were set up. 1 mL of blood was taken from each tube and centrifuged, and then dispersed with the above dispersion media respectively, placed in a shaker at 37 °C for incubation for 4 h, centrifuged at 3000 rpm for 10 min, and 100 μL of the supernatant was taken to measure the absorbance at 576 nm. Hemolysis rate (HR%) = (A sample - A background - A normal saline) / (A water - A normal saline) × 100%.

[0120] (2) Mouse body weight

[0121] Measure the body weights of mice in each group in the tumor-bearing mouse model.

[0122] (3) Indicators of the metabolic organ kidney

[0123] Collect the plasma of various mice in the tumor-bearing mouse model and measure it according to the CRE and BUN detection kits. Take kidney tissues for HE staining to observe their morphological changes.

[0124] Figure 8 For the hemolysis experiment (A) of the blood biocompatibility of the nanoparticles, the effect on body weight (B), and the effect on the main metabolic organ kidney (C, D), the above experiments show that: the nanoparticles have good safety performance, better tissue compatibility, the effect on body weight can be restored in the subsequent time, and they have good protection for the kidney.

[0125] In summary, the Pt-TPNs / siRNA nanoparticles prepared by the typical method of the present invention have the advantages of longer drug efficacy time, good drug efficacy, and low toxicity compared with the hydration therapy:

[0126] Compared with the hydration therapy, the Pt-TPNs / siRNA nanoparticles have the characteristics of larger particle size and are not easily filtered through the kidney, thus prolonging the residence time of the drug in the body, bringing longer drug efficacy and lower toxicity. The traditional hydration therapy promotes the excretion of drugs such as cisplatin by giving fluid replacement, magnesium supplementation, and mannitol diuresis, thereby reducing its residence time in the body. However, this method often requires frequent fluid replacement and diuretic treatments, which are not only time-consuming and laborious but also may cause other adverse reactions. In contrast, due to their larger particle size, the Pt-TPNs / siRNA nanoparticles often cannot easily pass through the filtration pore diameter of the glomerulus, resulting in a longer retention time of the drug in the body. Such characteristics enable the drug to exert its efficacy more fully and further improve the treatment effect.

[0127] The developed drug Pt-TPNs / siRNA nanoparticles can specifically knock out drug-resistant genes and increase the sensitivity of osteosarcoma cell lines to cisplatin, showing a sensitizing effect.

[0128] The abnormal expression of genes related to cisplatin resistance can lead to cisplatin resistance in osteosarcoma cells, thus limiting the therapeutic effect of cisplatin. To overcome this problem, the research and development team designed and synthesized Pt-TPNs / siRNA nanoparticles. These nanoparticles specifically target drug-resistant genes and introduce siRNA into osteosarcoma cells, thereby causing the expression of drug-resistant genes to be knocked out. The experimental results show that Pt-TPNs / siRNA nanoparticles successfully inhibit the expression of drug-resistant genes and further increase the sensitivity of osteosarcoma cells to cisplatin. The findings of this study indicate that by specifically knocking out genes related to cisplatin resistance, Pt-TPNs / siRNA nanoparticles can significantly increase the sensitivity of osteosarcoma cell lines to cisplatin, thereby achieving the effect of chemotherapy sensitization.

[0129] Example 7

[0130] 1. Effect of physically mixing TPNs with cisplatin

[0131] The HK-2 cells were passaged, the cells were counted so that the number of cells per well reached 1×105, seeded in a 12-well plate, the culture medium was gently blown with a tip to evenly distribute the cells, and then placed in an incubator for culture. After the cells adhered and grew for 24 h, the supernatant was discarded, and 500 μL of DMEM / F12 complete medium, complete medium containing 12 μM cisplatin, and complete medium containing 12 μM cisplatin and 100 μg / mL TPNs were added respectively. After drug treatment for 24 h, the medium was discarded, and the cells were washed three times with PBS. Then, 3 kinds of free radical probes, DCFH-DA, DHE and DAF-DA (diluted with phenol red-free DMEM / F12 containing 10% FBS at concentrations of 10 μM DCFH-DA, 5 μM DHE, and 5 μM DAF-DA respectively) were added, and the cells were incubated in an incubator for 30 min. The supernatant was discarded, and the cells were washed three times with PBS. Then, 500 μL of phenol red-free DMEM / F12 (10% FBS) was added, and fluorescence microscopy was used to take pictures.

DCFH-DA represents Dichlorodihydrofluorescein diacetate, a fluorescent probe used to detect intracellular oxidative stress. DHE represents Dihydroethidium, a fluorescent probe used to detect the production of reactive oxygen species (ROS) in cells. DAF-DA represents Diaminofluorescein diacetate, a fluorescent probe used to detect the production of nitric oxide (NO).

Claims

1. A pharmaceutically active phenolic chemically modified cisplatin material, characterized in that, is at least one compound having the general formula of Structure 1; In the said Formula 1, R1 is alkylene, substituted alkylene, alkenylene, arylene or cycloalkylene; R2 is an esterification residue of a pharmacologically active phenol; R3 is H or a substituent of Formula A; 2. The pharmaceutically active phenolic chemically modified cisplatin material according to claim 1, wherein R1 is C1-C6 alkylene or substituted alkylene with substituents on its carbon chain; The substituent is at least one of alkyl, alkoxy, halogen, phenyl, nitro, alkenyl; Preferably, R1 is C2-C4 alkylene.

3. The pharmaceutically active phenolic chemically modified cisplatin material according to claim 1, wherein The pharmacologically active phenol is a phenolic substance having at least one of anti-inflammatory, anti-tumor, antioxidant pharmacologically active properties, preferably at least one of tea polyphenols, catechins, catechin-3-gallol, catechin-3-gallate, gallic acid, caffeic acid, flavanone, flavon, anthocyanin; 4. A method for preparing a pharmaceutically active phenolic chemically modified cisplatin material according to any one of claims 1 to 3, characterized in that, It is obtained by subjecting cisplatin to hydroxylation treatment, then carrying out esterification carboxylation treatment with a carboxylation raw material, and then carrying out esterification treatment with a pharmacologically active phenol; The carboxylation raw material is at least one compound having Structures 2 and 3; In Formula 2, R4 and R5 are each independently at least one of halogen and hydroxyl.

5. The preparation method of the pharmacologically active phenolic chemically modified cisplatin material according to claim 4, characterized in that, Mix cisplatin and an oxidant for hydroxylation treatment; Preferably, the oxidant is hydrogen peroxide; Preferably, the conditions for the hydroxylation reaction are 30-60 °C; Preferably, the molar ratio of the product of the hydroxylation treatment to the carboxylation raw material is 1:2-3; Preferably, the temperature in the esterification carboxylation treatment stage is 10-40 °C; Preferably, the molar ratio of the product of the esterification carboxylation to the pharmacologically active phenol is 1:2-3.

6. Use of the pharmacologically active phenolic chemically modified cisplatin material according to any one of claims 1 to 3, characterized in that, Use it as a carrier for composite pharmacodynamic auxiliary components, and then prepare an anti-tumor drug; Preferably, the pharmacodynamic auxiliary components include at least one of anti-tumor active components, drug sensitivity components, and targeting aids; More preferably, the drug sensitivity components include drug sensitivity nucleic acids.

7. A nucleic acid-active phenolic chemically modified cisplatin composite anti-tumor active ingredient, characterized in that, It includes a nanocage assembled from a pharmacologically active phenol chemically modified cisplatin material as claimed in any one of claims 1-3 and nucleic acid filled inside it; Preferably, the nucleic acid is a sensitizing nucleic acid capable of increasing the anti-cancer activity of cisplatin; Preferably, the nucleic acid is complexed with the active phenol chemically modified cisplatin through the action of van der Waals forces, hydrogen bonds and chemical bonding; Preferably, in the nucleic acid-active phenol chemically modified cisplatin composite anti-tumor active component, the encapsulation rate is greater than 70%; 8. A method for preparing the nucleic acid-active phenolic chemically modified cisplatin composite anti-tumor active ingredient according to claim 7, characterized in that, It is obtained by self-assembling the pharmacologically active phenol chemically modified cisplatin material and nucleic acid.

9. Use of the nucleic acid-active phenol chemically modified cisplatin composite anti-tumor active component as claimed in claim 7 in the preparation of an anti-tumor drug.

10. An anti-tumor drug, characterized in that, Contains a pharmaceutically effective amount of the nucleic acid-active phenol chemically modified cisplatin composite anti-tumor active component as claimed in claim 7; Preferably, it also contains pharmaceutically acceptable excipients; Preferably, it has a pharmaceutically acceptable dosage form.

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