Anti-tumor compound as well as preparation method and application thereof
By preparing a ferrocene-nanodiamond and TAT-PEG-PLGA complex and combining it with doxorubicin, the anti-tumor effect of nanodiamond was enhanced, solving the problem that nanodiamond as a drug carrier could not kill tumor cells, and providing a new treatment method for tumors.
Patent Information
- Application Number
- CN202511146081.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-15
- Publication Date
- 2025-10-31
AI Technical Summary
In existing technologies, nanodiamonds as drug carriers cannot fully exert their anti-tumor properties, and traditional cancer treatment methods have problems such as high risk, large side effects, and tumor drug resistance.
An antitumor complex was prepared by coupling ferrocene with nanodiamond and combining it with TAT-PEG-PLGA to form a complex. Ferrocene was used to enhance the phospholipid free radical chain reaction, and doxorubicin was used to enhance the Fenton reaction.
This study achieved highly efficient killing effects of nanodiamonds in tumor cells, enhanced the targeting and cell penetration capabilities of anti-tumor drugs, and provided a new tumor treatment strategy.
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Figure CN120859945A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to an antitumor complex, its preparation method, and its application. Background Technology
[0002] Cancer is one of the most serious public health threats worldwide. Traditional clinical treatments for cancer mainly include surgical resection, radiotherapy, and chemotherapy. These traditional treatments have many problems; for example, surgery carries high risks, radiotherapy can cause significant adverse reactions and easily damage normal cells, and chemotherapy can easily lead to tumor drug resistance.
[0003] Nanodiamonds, as stable carbon materials, possess excellent biocompatibility, making them less effective at killing tumor cells. Current research primarily utilizes diamonds as drug carriers, without exploring the use of nanodiamonds' inherent anti-tumor properties in conjunction with drugs to achieve better therapeutic effects. Therefore, there is an urgent need to develop an anti-tumor drug that can fully utilize nanodiamonds. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide an anti-tumor complex, which is obtained by adsorbing ferrocene-nanodiamond and anti-tumor drugs by TAT-PEG-PLGA, and can be used to prepare anti-tumor drugs.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] In a first aspect, the present invention provides an antitumor complex comprising the following components in parts by weight: 10 parts of TAT-PEG-PLGA, 1 part of ferrocene-nanodiamond, and 0.2-0.5 parts of an antitumor drug.
[0007] The ferrocene-nanodiamond is a composite material of ferrocene and nanodiamond coupled together.
[0008] The ferrocene-nanodiamond and antitumor drugs are adsorbed onto the TAT-PEG-PLGA.
[0009] Ferroptosis is a novel form of cell death, primarily occurring through the generation of hydroxyl radicals in cells via a Fenton reaction, mediated by iron atoms. This invention has revealed that lipid peroxidation may play a crucial role in ferroptosis. This is because phospholipid chains contain polyunsaturated double bonds; these unsaturated fatty acids, under the influence of free radicals, generate phospholipid free radicals, which further attack phospholipids, triggering a chain reaction. Consequently, phospholipids are continuously oxidized and degraded, ultimately leading to cell membrane rupture and apoptosis. Simultaneously, lipid peroxides (LOOH) formed during lipid oxidation can also undergo a Fenton-like reaction with ferrous iron, generating new free radicals that further promote ferroptosis. The inventors, through the bioactivity of nanodiamonds in tumor cells, discovered that nanodiamonds are involved in the chain reaction induced by phospholipid free radicals. However, nanodiamonds, when used alone as antitumor drugs or drug delivery carriers, exhibit high biocompatibility, indicating that they cannot kill tumor cells through the chain reaction induced by phospholipid free radicals. Therefore, this invention aims to enhance phospholipid oxidative degradation and the ferroptosis process. Through drug screening, this invention discovered that ferrocene with an aromatic ring structure can effectively bind to nanodiamonds, which in principle can amplify the chain reaction of phospholipid free radicals and the ferroptosis process. Therefore, the combination of the two can exhibit good anti-tumor effects.
[0010] In this invention, TAT, or cell-penetrating peptide, has the sequence GRKKRRQRRRPQ; PEG refers to polyethylene glycol; and PLGA is polylactic-co-glycolic acid copolymer. In the TAT-PEG-PLGA structure, TAT is linked to PLGA via PEG. PLGA, a widely used biodegradable polymer, is renowned for its excellent biocompatibility and degradability. As the backbone of nanoparticles, it not only provides high mechanical strength but also has the function of regulating drug release. PEG, as a hydrophilic polymer, can enhance the water solubility and biocompatibility of nanoparticles. PEG-modified nanoparticles have "stealth" properties, which helps them evade immune clearance, promote the accumulation of nanoparticles at tumor sites, and prolong their residence time in the bloodstream. In addition, PEG can reduce the non-specific adsorption of nanoparticles in vivo, enhancing their stability and targeting. TAT, as the transduction domain peptide sequence of HIV-1 virus, is known for its efficient membrane-penetrating ability, and its main function is to improve the uptake efficiency of nanoparticles by cells, especially for cells or tissues that are difficult to penetrate. Therefore, TAT-PEG-PLGA possesses high membrane penetration ability, water solubility, biocompatibility, stability, and targeting properties; furthermore, the vesicles formed by TAT-PEG-PLGA exhibit adsorption capacity. This invention utilizes TAT-PEG-PLGA to load ferrocene-nanodiamond and doxorubicin, enabling both to effectively enter tumor cells and exert anti-tumor effects. Doxorubicin can increase the intracellular concentration of hydrogen peroxide, a substrate for the Fenton reaction, thus further enhancing ferroptosis and strengthening the anti-tumor efficacy.
[0011] Preferably, the antitumor drug includes doxorubicin.
[0012] More preferably, the antitumor complex comprises the following components in parts by weight: 2 parts TAT-PEG-PLGA, 1 part ferrocene-nanodiamond, and 0.2-0.5 parts antitumor drug.
[0013] Preferably, the ferrocene-nanodiamond is prepared by the following method:
[0014] (1) After the nanodiamonds are mixed with a mixed acid and reacted evenly, they are then mixed with an alkali to obtain carboxylated nanodiamonds.
[0015] (2) Carboxylated nanodiamonds and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were mixed evenly in an organic solvent to obtain mixture a;
[0016] (3) Add aminoferrocene and N-hydroxysuccinimide to the mixture a obtained in step (2) and react to obtain the ferrocene-nanodiamond.
[0017] This invention first utilizes a mixed acid reaction with nanodiamonds. The mixed acid provides stronger oxidizing power than a single acid. For example, this invention uses sulfuric acid and nitric acid. Sulfuric acid provides a strong acid environment, while nitric acid provides oxidizing power, causing oxygen-containing functional groups, such as carboxyl groups, to form on the surface of the diamond. Then, a base is added to stop the reaction from continuing due to residual strong acid. This reaction yields carboxylated diamond, which can undergo an amidation reaction with aminoferrocene to ultimately synthesize ferrocene-nanodiamonds.
[0018] Preferably, the mixed acid in step (1) includes sulfuric acid and nitric acid.
[0019] More preferably, the volume ratio of the sulfuric acid and nitric acid mixture is sulfuric acid:nitric acid = (5-6):1.
[0020] Preferably, the alkali in step (1) includes hydrogen peroxide.
[0021] Preferably, the organic solvent in step (2) includes dimethyl sulfoxide.
[0022] Preferably, in step (1), the reaction conditions for uniformly mixing nanodiamonds and mixed acid are: reacting at 60-80℃ for 12-24 hours.
[0023] More preferably, in step (1), the reaction conditions for uniformly mixing nanodiamonds and mixed acid are: reacting at 70°C for 24 hours.
[0024] Preferably, in step (1), the reaction conditions with the alkali are: 60-80℃ for 1.5-2 hours.
[0025] More preferably, in step (1), the reaction conditions with the alkali are: 70°C for 1.5-2 hours.
[0026] Preferably, in step (2), the reaction conditions are: reaction at 20-25°C for 1-2.5 hours.
[0027] More preferably, in step (2), the reaction conditions are: reaction at 25°C for 1.5 hours.
[0028] Preferably, in step (3), the mass ratio of mixture a, aminoferrocene and N-hydroxysuccinimide is mixture a: aminoferrocene: N-hydroxysuccinimide = (20-30): (0.1-0.5): (20-30).
[0029] Preferably, in step (3), the reaction conditions are: reaction at 20-25°C for 10-24 hours.
[0030] More preferably, in step (3), the reaction conditions are: reaction at 25°C for 24 hours.
[0031] Secondly, the present invention provides a method for preparing the aforementioned antitumor complex, comprising the following steps:
[0032] S1. Ferrocene-nanodiamond and TAT-PEG-PLGA are mixed evenly in an organic solvent to obtain an oil phase;
[0033] S2. Dissolve the antitumor drug in an aqueous solvent to obtain an aqueous phase;
[0034] S3. Mix the oil phase and the aqueous phase evenly, separate them, and obtain the antitumor complex.
[0035] TAT-PEG-PLGA is an organic polymer; ferrocene-nanodiamond has hydrophobic groups on its surface, making it miscible with TAT-PEG-PLGA in organic solvents. In the aqueous phase, the two can self-assemble to form nanomicelles. This invention uses dichloromethane as the organic solvent, which is volatile and gradually evaporates at room temperature as nanomicelles form.
[0036] Preferably, the organic solvent in step S1 includes dichloromethane.
[0037] Preferably, the aqueous solvent in step S2 includes a phosphate buffer solution.
[0038] Preferably, in step S3, the oil phase and the water phase are mixed by ultrasonication.
[0039] Preferably, the conditions for ultrasonic mixing are: ultrasonication at 40-50 kHz for 10-20 minutes.
[0040] Preferably, in step S3, the separation conditions are: centrifugation at 5000-6000 rpm for 20-30 minutes.
[0041] Preferably, in step S3, the volume ratio of the oil phase to the water phase is oil phase:water phase = 1:(5-15).
[0042] Thirdly, the present invention provides the application of the aforementioned antitumor complex in the preparation of antitumor drugs.
[0043] The beneficial effects of this invention are as follows:
[0044] Nanodiamonds, despite their good biocompatibility, cannot effectively kill tumor cells. This invention combines ferrocene, which has an aromatic ring structure, with nanodiamonds to fully exert a good anti-tumor effect. TAT-PEG-PLGA has high membrane penetration ability, water solubility, biocompatibility, stability, and targeting. This invention utilizes TAT-PEG-PLGA with surface adsorption capacity to load ferrocene-nanodiamonds and doxorubicin to obtain an anti-tumor complex, enabling both to effectively enter tumor cells and exert a synergistic anti-tumor effect. The anti-tumor complex obtained by this invention can effectively inhibit the activity of melanoma cells A375, and is an effective tumor inhibitor, providing a new treatment strategy and option for tumor treatment. Attached Figure Description
[0045] Figure 1 This is an electron micrograph of the antitumor complex.
[0046] Figure 2 The results of A375 viability assays for melanoma cells after different group treatments. Detailed Implementation
[0047] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0048] Unless otherwise specified, the experimental methods used in this invention are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.
[0049] Example 1:
[0050] An embodiment of the antitumor complex of the present invention; the antitumor complex comprises the following components in parts by weight: 2 parts TAT-PEG-PLGA, 1 part ferrocene-nanodiamond, and 0.2 parts doxorubicin.
[0051] The ferrocene-nanodiamond is a composite material of ferrocene and nanodiamond coupled together.
[0052] The ferrocene-nanodiamond and doxorubicin are adsorbed onto the TAT-PEG-PLGA.
[0053] The preparation method of the antitumor complex includes the following steps:
[0054] A. Preparation of ferrocene-nanodiamond:
[0055] (1) Nanodiamonds were mixed with a mixture of sulfuric acid and nitric acid (volume ratio of sulfuric acid: nitric acid = 5:1) and reacted at 70°C for 24 hours to obtain product one; product one was mixed with hydrogen peroxide and reacted at 70°C for 1.5 hours to obtain carboxylated nanodiamonds.
[0056] (2) Carboxylated nanodiamonds and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were mixed evenly in dimethyl sulfoxide and reacted at 25°C for 1.5 hours to obtain mixture a;
[0057] (3) Add 0.1 part of aminoferrocene and 20 parts of N-hydroxysuccinimide to the 20 parts of mixture a obtained in step (2), and react at 25°C for 24 hours to obtain ferrocene-nanodiamond;
[0058] B. Preparation of antitumor complex:
[0059] (4) Ferrocene-nanodiamond and TAT-PEG-PLGA were mixed evenly in dichloromethane to obtain an oil phase;
[0060] (5) Dissolve doxorubicin in a phosphate buffer solution to obtain an aqueous phase;
[0061] (6) The oil phase and the water phase were mixed evenly by ultrasonication at a volume ratio of 1:10 (ultrasonication at 40 kHz for 20 minutes), and centrifuged at 5000 rpm for 30 minutes to obtain the antitumor complex.
[0062] Example 2:
[0063] An embodiment of the antitumor complex of the present invention; the antitumor complex comprises the following components in parts by weight: 10 parts of TAT-PEG-PLGA, 1 part of ferrocene-nanodiamond, and 0.5 parts of doxorubicin.
[0064] The ferrocene-nanodiamond is a composite material of ferrocene and nanodiamond coupled together.
[0065] The ferrocene-nanodiamond and doxorubicin are adsorbed onto the TAT-PEG-PLGA.
[0066] The preparation method of the antitumor complex includes the following steps:
[0067] A. Preparation of ferrocene-nanodiamond:
[0068] (1) Nanodiamonds were mixed with a mixture of sulfuric acid and nitric acid (volume ratio of sulfuric acid: nitric acid = 6:1) and reacted at 80°C for 12 hours to obtain product one; product one was mixed with hydrogen peroxide and reacted at 80°C for 1.5 hours to obtain carboxylated nanodiamonds.
[0069] (2) Carboxylated nanodiamonds and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were mixed evenly in dimethyl sulfoxide and reacted at 25°C for 1 hour to obtain mixture a;
[0070] (3) Add 0.5 parts of aminoferrocene and 30 parts of N-hydroxysuccinimide to the 30 parts of mixture a obtained in step (2), and react at 20°C for 24 hours to obtain ferrocene-nanodiamond;
[0071] B. Preparation of antitumor complex:
[0072] (4) Ferrocene-nanodiamond and TAT-PEG-PLGA were mixed evenly in dichloromethane to obtain an oil phase;
[0073] (5) Dissolve doxorubicin in a phosphate buffer solution to obtain an aqueous phase;
[0074] (6) The oil phase and the water phase were mixed evenly by ultrasonication at a volume ratio of 1:5 (sonicated at 50 kHz for 10 minutes), and centrifuged at 6000 rpm for 20 minutes to obtain the antitumor complex.
[0075] Example 3:
[0076] An embodiment of the antitumor complex of the present invention; the antitumor complex comprises the following components in parts by weight: 5 parts of TAT-PEG-PLGA, 1 part of ferrocene-nanodiamond, and 0.3 parts of doxorubicin.
[0077] The ferrocene-nanodiamond is a composite material of ferrocene and nanodiamond coupled together.
[0078] The ferrocene-nanodiamond and doxorubicin are adsorbed onto the TAT-PEG-PLGA.
[0079] The preparation method of the antitumor complex includes the following steps:
[0080] A. Preparation of ferrocene-nanodiamond:
[0081] (1) Nanodiamonds were mixed with a mixture of sulfuric acid and nitric acid (volume ratio of sulfuric acid: nitric acid = 5:1) and reacted at 60°C for 18 hours to obtain product one; product one was mixed with hydrogen peroxide and reacted at 60°C for 2 hours to obtain carboxylated nanodiamonds.
[0082] (2) Carboxylated nanodiamonds and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were mixed evenly in dimethyl sulfoxide and reacted at 20°C for 2.5 hours to obtain mixture a;
[0083] (3) Add 0.3 parts of aminoferrocene and 25 parts of N-hydroxysuccinimide to the 25 parts of mixture a obtained in step (2), and react at 25°C for 10 hours to obtain ferrocene-nanodiamond;
[0084] B. Preparation of antitumor complex:
[0085] (4) Ferrocene-nanodiamond and TAT-PEG-PLGA were mixed evenly in dichloromethane to obtain an oil phase;
[0086] (5) Dissolve doxorubicin in a phosphate buffer solution to obtain an aqueous phase;
[0087] (6) The oil phase and the water phase were mixed evenly by ultrasonication at a volume ratio of 1:15 (sonicated at 45 kHz for 15 minutes), and centrifuged at 5000 rpm for 25 minutes to obtain the antitumor complex.
[0088] Test Example 1: TEM Analysis
[0089] The antitumor complexes obtained in Examples 1-3 were examined microscopically using transmission electron microscopy (TEM), and the results are as follows: Figure 1 As shown (with Example 1 as an example).
[0090] The electron microscope image shows that TAT-PEG-PLGA forms vesicles (white spheres) with ferrocene-nanodiamond (black particles) and doxorubicin adsorbed on the outside.
[0091] Test Example 2: Effect of antitumor complex on the viability of melanoma cells A375
[0092] Melanoma cells A375 were cultured in 1640 medium containing 10% fetal bovine serum and placed in a constant temperature incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were cultured in 96-well plates. When the cell density reached 80%-90%, different reagents were added to treat different groups.
[0093] Group 1: Control group: Phosphate buffer solution;
[0094] Group 2: Ferrocene-nanodiamond with concentrations of 2.5 μg / mL and 5 μg / mL were added;
[0095] Group 3: Doxorubicin at concentrations of 2.5 μg / mL and 5 μg / mL was added;
[0096] Group 4: Antitumor complexes were added at concentrations of 2.5 μg / mL and 5 μg / mL (Example 1).
[0097] Measure the absorbance of each group and calculate cell viability according to the following formula:
[0098] Cell viability (%) = (Experimental group absorbance / Control group absorbance) * 100%
[0099] The results are as follows Figure 2 As shown, the antitumor complex prepared in this invention can effectively inhibit the activity of melanoma cells A375, and is an effective tumor inhibitor.
[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. An antitumor complex, characterized in that, The antitumor complex comprises the following components in parts by weight: 2-10 parts TAT-PEG-PLGA, 1 part ferrocene-nanodiamond, and 0.2-0.5 parts antitumor drug; The ferrocene-nanodiamond is a composite material of ferrocene and nanodiamond coupled together. The ferrocene-nanodiamond and antitumor drugs are adsorbed onto the TAT-PEG-PLGA.
2. The antitumor complex according to claim 1, characterized in that, The antitumor drugs include doxorubicin.
3. The antitumor complex according to claim 1, characterized in that, The ferrocene-nanodiamond was prepared by the following method: (1) After the nanodiamonds are mixed with a mixed acid and reacted evenly, they are then mixed with an alkali to obtain carboxylated nanodiamonds. (2) Carboxylated nanodiamonds and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were mixed evenly in an organic solvent to obtain mixture a; (3) Add aminoferrocene and N-hydroxysuccinimide to the mixture a obtained in step (2) and react to obtain the ferrocene-nanodiamond.
4. The antitumor complex according to claim 3, characterized in that, The mixed acid in step (1) includes sulfuric acid and nitric acid; And / or, the base in step (1) includes hydrogen peroxide.
5. The antitumor complex according to claim 3, characterized in that, The organic solvent in step (2) includes dimethyl sulfoxide.
6. A method for preparing an antitumor complex according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Ferrocene-nanodiamond and TAT-PEG-PLGA are mixed evenly in an organic solvent to obtain an oil phase; S2. Dissolve the antitumor drug in an aqueous solvent to obtain an aqueous phase; S3. Mix the oil phase and the aqueous phase evenly, separate them, and obtain the antitumor complex.
7. The method for preparing the antitumor complex according to claim 6, characterized in that, The organic solvent in step S1 includes dichloromethane; And / or, the aqueous solvent in step S2 includes a phosphate buffer solution.
8. The method for preparing the antitumor complex according to claim 6, characterized in that, In step S3, the oil phase and the water phase are mixed by ultrasonication.
9. The method for preparing the antitumor complex according to claim 6, characterized in that, In step S3, the volume ratio of the oil phase to the water phase is oil phase:water phase = 1:(5-15).
10. The use of the antitumor complex according to any one of claims 1-5 in the preparation of an antitumor drug.