WO2024040728A1

WO2024040728A1 - Platinum-based drug carbon nanodot, and method for preparing same and carbon nanodot protein compound and use thereof

Info

Publication number: WO2024040728A1
Application number: PCT/CN2022/126494
Authority: WO
Inventors: 曲松楠; 郭东波; 汤子康
Original assignee: 澳门大学
Priority date: 2022-08-25
Filing date: 2022-10-20
Publication date: 2024-02-29
Also published as: CN115364215A; CN115364215B

Abstract

Provided are a platinum-based drug carbon nanodot and a method for preparing same and a carbon nanodot protein compound and use thereof. The platinum-based drug carbon nanodot comprises a carbon-based core having the characteristic of visible light absorption. The carbon-based core is combined with a tetravalent platinum element via a coordinate bond, and the platinum-based drug carbon nanodot can be reduced under light irradiation conditions to obtain a divalent platinum compound and a hydroxyl radical. The method for preparing the platinum-based drug carbon nanodot comprises subjecting a disubstituted aromatic compound and a tetravalent platinum compound to a solvothermal reaction to obtain the platinum-based drug carbon nanodot. Under irradiation of visible light, the carbon-based core generates hole-electron pairs. Excited-state electrons are captured by platinum (IV) to undergo a reduction reaction, which rapidly generates a Pt (II) compound with high cytotoxicity and a hydroxyl radical and reduces the pH value of cells and a tumor site, resulting in immunogenic death of cancer cells and activation of the anti-tumor immune response of the body as well while killing the cancer cells. The platinum-based drug carbon nanodot and the protein compound thereof of the present invention can serve as a high-efficiency photoactive anti-cancer drug for precision treatment of tumors.

Description

Platinum-medicated carbon nanodots and preparation methods thereof, carbon nanodot protein complexes and applications

Cross-references to related applications

This disclosure requires priority for the Chinese patent application with the application number CN202211027234.6 and the name "Platinum Drug Carbon Nanodots and Preparation Methods, Carbon Nanodot Protein Complexes and Applications" submitted to the China Patent Office on August 25, 2022. rights, the entire contents of which are incorporated into this disclosure by reference.

Technical field

The present disclosure relates to the technical field of platinum anticancer drugs, specifically, to a platinum drug carbon nanodot and its preparation method, carbon nanodot protein complex and application.

Background technique

Traditional chemotherapy drugs such as cisplatin and other divalent platinum complexes are a type of metal complex with anti-cancer activity. They were widely used after B. Rosenborg et al. first discovered in 1965 that they could inhibit the growth of tumor cells. It plays an important role in cancer chemotherapy and plays an important role in chemotherapy drugs (Nature1965, 205, 698.). After that, anti-cancer drugs such as carboplatin, oxaliplatin and other divalent platinum complexes were approved for marketing. However, general platinum complexes are ineffective when taken orally. Most of the clinical use methods are intravenous drip administration. After intravenous injection, cisplatin quickly disappears in the plasma and is rapidly distributed throughout the body, especially in the liver, kidneys, and large and small intestines. It is most distributed in the skin and skin, so it has serious side effects, such as causing nephrotoxicity, bone marrow suppression and gastrointestinal side effects (Chem. Rev. 2014, 114, 4470-4495). At the same time, platinum complexes such as cisplatin have a short half-life in the blood, so the proportion of them reaching the lesion is very low and the efficacy is poor.

Photocontrolled release of platinum-based drugs is a personalized medical method that uses a specific lighting environment to accurately release the contained drug molecules at the lesion in time and space. It has many advantages such as high drug utilization rate and low toxic and side effects. The advantages provide new ideas for the precise treatment of various major diseases, such as tumors. Traditional light-controlled drug release systems are divided into two methods: physical loading and chemical bonding. The physical packaging method has problems such as low drug loading rate and drug leakage during transportation, which limits its further application. This situation can be effectively overcome through covalent bonding. However, the current photocontrollable platinum drugs are mainly small molecule compounds, which have the disadvantages of poor water solubility, poor tumor enrichment ability, and short circulation time (Nat. Rev. Cancer. 3 (2003) 380-387).

Contents of the invention

The present disclosure provides a platinum-medicated carbon nanodot, which includes a carbon-based core with visible light absorption properties. The carbon-based core is coordinated and combined with a tetravalent platinum element. The platinum-medicated carbon nanodot can be reduced to divalent platinum under light irradiation conditions. compounds and hydroxyl radicals.

Optionally, the light irradiation wavelength is 200nm-1200nm.

The present disclosure provides a method for preparing platinum-medicated carbon nanodots, which includes performing a solvothermal reaction between a disubstituted aromatic compound and a tetravalent platinum complex to obtain platinum-medicated carbon nanodots.

Optionally, the disubstituted aromatic compound is a disubstituted compound of benzene, naphthalene, anthracene or phenanthrene; and/or, the tetravalent platinum complex is selected from at least one of oxidized cisplatin and diacid cisplatin. , the chemical formula of the oxidized cisplatin is

Optionally, the disubstituted aromatic compound is an aromatic compound of diamine.

Alternatively, the disubstituted aromatic compound is a compound represented by formula (I) or formula (II);

In formula (I) and formula (II), R ₁ and R ₂ are each independently selected from -(CH ₂ ) _m NH ₂ , -O(CH ₂ ) _n NH ₂ , -(CH ₂ ) _m OH, -O Any one of (CH ₂ ) _n OH, -(CH ₂ ) _m NO ₂ , -O(CH ₂ ) _n NO ₂ , -(CH ₂ ) _m COOH, -O(CH ₂ ) _n COOH; m and n takes 0 to 10 respectively.

Alternatively, R ₁ and R ₂ are each independently selected from any one of -NH ₂ , -ONH ₂ , -OH, -NO _{2 ,} -ONO ₂ , -COOH, and -OCOOH; and/or, tetravalent The platinum complex is cisplatin oxide.

Alternatively, the disubstituted aromatic compound is at least one of the following compounds:

Alternatively, the disubstituted aromatic compound is

Optionally, the molar ratio of the disubstituted aromatic compound to the tetravalent platinum complex is 1-1000:1-1000.

Alternatively, the reaction solvent is selected from at least one of dimethyl sulfoxide, N, N'-dimethylformamide, water, formic acid, ethanol, diethyl ether, tetrahydrofuran, folic acid and acetone;

And/or, the ratio of the disubstituted aromatic compound to the solvent is 0.001g/mL～1g/mL.

Optionally, the temperature of the solvothermal reaction is 100-250°C, and the reaction time is 1-10 hours.

The present disclosure also provides a carbon nanodot protein complex, which is obtained by complexing the above-mentioned platinum-medicated carbon nanodots and macromolecular protein.

The present disclosure also provides a carbon nanodot protein complex, including the above-mentioned platinum-medicated carbon nanodots or the platinum-medicated carbon nanodots prepared by the above-mentioned preparation method, and macromolecular proteins.

Optionally, the absorption peak of the ultraviolet-visible absorption spectrum of the carbon nanodot protein complex is in the range of 380-800 nm.

Optionally, the fluorescence emission range of the carbon nanodot protein complex is 500-850 nm.

Optionally, the size of the carbon nanodot protein complex ranges from 10 to 500 nm.

The present disclosure also provides the use of the above-mentioned platinum-drug carbon nanodots or the platinum-drug carbon nanodots or carbon nanodot protein complexes prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

The present disclosure also provides the above-mentioned platinum-medicated carbon nanodots or the above-mentioned platinum-medicated carbon nanodots prepared by the above-mentioned preparation method or the above-mentioned carbon nanodot protein complex for use in treating tumors.

The present disclosure also provides a method for treating tumors, which method includes: administering the above-mentioned platinum-medicine carbon nanodots or the platinum-medicine carbon nanodots prepared by the above-mentioned preparation method or the above-mentioned carbon nanodots to a subject in need. protein complex.

Description of drawings

In order to explain the technical solutions of the embodiments of the present disclosure more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present disclosure and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic diagram of the illumination reaction of platinum-medicated carbon nanodots;

Figure 2 is a transmission electron microscope image of platinum-medicated carbon nanodots in Experimental Example 1;

Figure 3 is the EDS image of platinum-medicated carbon nanodots in Experimental Example 1;

Figure 4 is the XPS spectrum of platinum-medicated carbon nanodots in Experimental Example 1;

Figure 5 is the Pt 4f energy spectrum of platinum-medicated carbon nanodots in Experimental Example 1;

Figure 6 is the N 1s energy spectrum of platinum-medicated carbon nanodots in Experimental Example 2;

Figure 7 is the O 1s energy spectrum of platinum-medicated carbon nanodots in Experimental Example 2;

Figure 8 is the hydrogen spectrum NMR spectrum of platinum-medicated carbon nanodots in Experimental Example 2;

Figure 9 is the electron spin energy spectrum of platinum-medicated carbon nanodots in Experimental Example 2;

Figure 10 is a flow chart of platinum-medicated carbon quantum dot illumination in Experimental Example 3;

Figure 11 is a transmission electron microscope image of platinum-medicated carbon nanodots@BSA in Experimental Example 4;

Figure 12 shows the UV and fluorescence spectra of platinum-medicated carbon nanodots@BSA in Experimental Example 4;

Figure 13 shows the platinum drug sustained release curve of the platinum drug carbon nanodots in Experimental Example 5 under light or glutathione reducing environment;

Figure 14 is the pH change curve of the platinum-medicated carbon quantum dots under light in Experimental Example 6;

Figure 15 shows the intracellular platinum content of platinum-medicated carbon quantum dots and platinum-medicated carbon nanodots@BSA at different incubation times in Experimental Example 7;

Figure 16 is a fluorescence microscope picture of the platinum-medicated carbon quantum dots and platinum-medicated carbon nanodots@BSA in Experimental Example 7 after they were co-incubated in 4T1 cells for 4 hours;

Figure 17 is a diagram showing the inhibitory effect of platinum-medicated carbon nanodots on 4T1 breast cancer cells in Experimental Example 8;

Figure 18 is a diagram showing the inhibitory effect of platinum drug carbon nanodots@BSA on 4T1 breast cancer cells in Experimental Example 8;

Figure 19 is the molecular imprinting diagram of phosphorylated histone H2AX (p-H2A.

Figure 20 shows the effects of platinum-medicated carbon quantum dots and platinum-medicated carbon quantum dots@BSA on the expression of reactive oxygen species in 4T1 breast cancer cells before and after irradiation in Experimental Example 9;

Figure 21 is a flow cytometry diagram of the apoptosis effect of platinum-medicated carbon quantum dots and platinum-medicated carbon quantum dots@BSA on 4T1 breast cancer cells under Fer-1 inhibition before and after irradiation in Experimental Example 9;

Figure 22 shows the immunofluorescence of calreticulin (CRT) and high mobility group protein B1 (HMGB1) in 4T1 breast cancer cells after irradiation with cisplatin, platinum-drug carbon quantum dots, and platinum-drug carbon quantum dots@BSA in Experimental Example 10. picture;

Figure 23 is a schematic diagram of the inhibitory effect of platinum-medicated carbon quantum dots on tumor growth in 4T1 mice before and after irradiation in Experimental Example 11;

Figure 24 is a schematic diagram of the inhibitory effect of platinum-medicated carbon quantum dots on distal tumor growth in 4T1 mice before and after irradiation in Experimental Example 11;

Figure 25 is the survival curve of 4T1 mice before and after irradiation with platinum drug carbon quantum dots in Experimental Example 12;

Figure 26 shows photos of lung metastasis in 4T1 mice before and after irradiation with platinum-medicine carbon quantum dots in Experimental Example 12.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments and examples of the present disclosure clearer, the technical solutions in the embodiments and examples of the present disclosure will be clearly and completely described below. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

The platinum-medicated carbon nanodots and their preparation methods, carbon nanodot protein complexes and applications provided by the present disclosure will be described below.

Some embodiments of the present disclosure provide a platinum-medicated carbon nanodot, which includes a carbon-based core chemically bonded with a tetravalent platinum element, the carbon-based core has visible light absorption, and the platinum-medicated carbon nanodots Under irradiation conditions, divalent platinum compounds and hydroxyl radicals can be obtained by reduction. Alternatively, the light irradiation wavelength is 200nm-1200nm, such as 300nm-1100nm, 400nm-1000nm or 500nm-900nm, such as 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1100nm or 1 200nm etc., or The interval value between any two of the above endpoint values.

Referring to Figure 1, under visible light irradiation, the carbon-based core generates hole-electron pairs, and the excited electrons are captured by platinum (Pt) (IV) to undergo a reduction reaction and release compounds containing platinum (Pt) (II); the valence band The holes formed by light excitation react with the hydroxyl ions in the aqueous solution to emit oxidation reactions on the surface of the nanoparticles, forming hydroxyl radicals and causing a decrease in the pH value of the aqueous solution.

Some embodiments of the present disclosure provide a method for preparing platinum-medicated carbon nanodots, which includes performing a solvothermal reaction on a disubstituted aromatic compound and a tetravalent platinum complex to obtain platinum-medicated carbon nanodots.

In some embodiments, the disubstituted aromatic compound is a disubstituted compound of benzene, naphthalene, anthracene or phenanthrene; and/or the tetravalent platinum complex is selected from at least one of oxidized cisplatin and diacid cisplatin, and the oxidized cisplatin is The chemical formula of platinum is

In some embodiments, the disubstituted aromatic compound is an aromatic compound of a diamine.

In some embodiments, the disubstituted aromatic compound is a compound represented by formula (I) or formula (II);

In formula (I) and formula (II), R ₁ and R ₂ are each independently selected from -(CH ₂ ) _m NH ₂ , -O(CH ₂ ) _n NH ₂ , -(CH ₂ ) _m OH, -O Any one of (CH ₂ ) _n OH, -(CH ₂ ) _m NO ₂ , -O(CH ₂ ) _n NO ₂ , -(CH ₂ ) _m COOH, -O(CH ₂ ) _n COOH; m and n ranges from 0 to 10 respectively, for example, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; for example, n is 0, 1, 2, 3, 4, 5, 6, 7 ,8,9 or 10.

For example, the reaction formula for preparing platinum-medicated carbon nanodots can be:

In some optional embodiments, R ₁ and R ₂ are each independently selected from any one of -NH ₂ , -ONH ₂ , -OH, -NO _{2 ,} -ONO ₂ , -COOH, and -OCOOH; and/ Alternatively, the tetravalent platinum complex is cisplatin oxide.

In some embodiments, the disubstituted aromatic compound is at least one of the following compounds:

In some embodiments, the disubstituted aromatic compound is

The reaction formula for preparing platinum-medicated carbon nanodots is:

During the above reaction process, the hydroxyl and amino groups of the oxidized cisplatin are coordinately combined to load the oxidized cisplatin on the carbon-based core. It should be noted that aromatic compounds of diamines include, but are not limited to, any of the following: o-phenylenediamine, p-phenylenediamine, m-phenylenediamine; and any two amino groups such as naphthalene, anthracene, and phenanthrene. Permutations.

In some embodiments, aromatic compounds of diamines include, but are not limited to, naphthalene, anthracene, and phenanthrene each having any two amine groups.

In some embodiments, in order to enable better formation of carbon nanodots, the molar ratio of the disubstituted aromatic compound and the tetravalent platinum complex is 1 to 1000:1 to 1000, such as 1 to 500:1 to 500, 1 to 400:1～400 or 1～200:1～200, optionally 1～100:1～100, optionally 1～3:1～3, such as 1:10, 1:9, 1:8 ,1:7,1:6,1:5,1:4,1:3,1:2,1:1,2:1,3:1,4:1,5:1,6:1,7 :1, 8:1, 9:1, 10:1, 2:3 or 3:2. In some embodiments, the molar ratio of the disubstituted aromatic compound to the tetravalent platinum complex may be 1:1.

Optionally, during the solvothermal reaction, in order to fully mix the reactants, the reaction process needs to be carried out in a solvent system. The reaction solvent used to dissolve the disubstituted aromatic compound and the tetravalent platinum complex includes but is not limited to At least one of dimethyl sulfoxide, N, N'-dimethylformamide, water, formic acid, ethanol, diethyl ether, tetrahydrofuran, folic acid and acetone. That is, the reaction solvent can be selected individually from dimethyl sulfoxide, N, N'-dimethylformamide, water, formic acid, ethanol, ether, tetrahydrofuran, folic acid or acetone, or two or more of these substances can be selected mixture, the mixing ratio is not limited (that is, mixed in any proportion).

Alternatively, in some embodiments, the ratio of the disubstituted aromatic compound to the reaction solvent is 0.001g/mL～1g/mL, such as 0.005g/mL～0.5g/mL, 0.01g/mL～0.1g/mL or 0.05 g/mL～0.08g/mL, such as 0.001g/mL, 0.002g/mL, 0.004g/mL, 0.008g/mL, 0.01g/mL, 0.02g/mL, 0.03g/mL, 0.04g/mL, 0.05g/mL, 0.06g/mL, 0.07g/mL, 0.08g/mL, 0.09g/mL, 0.1g/mL, 0.2g/mL, 0.3g/mL, 0.4g/mL, 0.5g/mL, 0.6g/mL, 0.7g/mL, 0.8g/mL, 0.9g/mL or 1g/mL, etc., or an interval value between any two of the above endpoint values.

Alternatively, in some embodiments, the temperature of the solvothermal reaction is 100-250°C, for example, it can be 120-220°C, 160-200°C, or 170-190°C, such as 100°C, 110°C, or 120°C. , 130℃, 140℃, 150℃, 160℃, 170℃, 180℃, 190℃, 200℃, 210℃, 220℃, 230℃, 240℃ or 250℃, etc., or between any two of the above endpoint values interval value; the reaction time is 1-10 hours, for example, it can be 1.5-9.5 hours, 3-8 hours or 4-6 hours, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours , 7 hours, 8 hours, 9 hours or 10 hours, etc., or an interval value between any two of the above endpoint values.

Some embodiments of the present disclosure also provide a carbon nanodot protein complex, which is obtained by complexing the above-mentioned platinum-medicated carbon nanodots and macromolecular protein.

In some embodiments, macromolecular proteins optionally include, but are not limited to, bovine serum albumin, fibrin, and collagen.

The carbon nanodot protein complex has high cell endocytosis efficiency and tumor enrichment efficiency. Cancer cells or tumors enriched with platinum-medicine carbon nanodots can quickly produce highly cytotoxic Pt(II) compounds and hydroxyl radicals under visible light, and down-regulate the pH value in cells and tumor sites, causing cancer Cells produce immunogenic death, killing cancer cells and activating the body's anti-tumor immune response.

Some embodiments of the present disclosure also provide the use of the above-mentioned platinum drug carbon nanodots or carbon nanodot protein complexes in the preparation of anti-tumor drugs.

Some embodiments of the present disclosure also provide the above-mentioned platinum-medicated carbon nanodots or the above-mentioned platinum-medicated carbon nanodots prepared by the above-mentioned preparation method or the above-mentioned carbon nanodot protein complex for use in treating tumors.

Some embodiments of the present disclosure also provide a method for treating tumors, which method includes: administering the above-mentioned platinum-medicine carbon nanodots or the platinum-medicine carbon nanodots prepared by the above preparation method or the above-mentioned carbon to a subject in need. Nanodot protein complex.

The platinum-medicine carbon nanodots and their protein complexes in the above embodiments can be used as highly efficient photoactive anti-cancer drugs for precise tumor treatment.

The disclosed platinum-medicine carbon nanodots and their protein complexes have controllable light-responsive release characteristics. Under the condition of light, they can be reduced to obtain divalent platinum anti-cancer drugs, strong tumor-killing hydroxyl radicals, and cause Acidification within tumor cells, therefore, not only reduces the side effects of platinum drugs, but also improves the anti-cancer effect of traditional cisplatin; compared with traditional single drug cisplatin, the platinum drug carbon nanodots have diverse forms, and their proteins After compounding, the circulation time in the body and the enrichment in the tumor site can be improved.

Example

The features and performance of the present disclosure will be described in further detail below with reference to examples.

Example 1

This embodiment provides a method for preparing platinum-medicated carbon nanodots, the operation of which is:

Weigh 0.1g cisplatin oxide and 0.1g o-phenylenediamine. Then add 30 mL of N, N-dimethylformamide to the polytetrafluoroethylene reactor, and then place it in an oven to react at 200°C for 6 hours. Then the solution was taken out, dialyzed, filtered, and freeze-dried to obtain a platinum-medicated carbon nanodot solid.

Example 2

Weigh 1g cisplatin oxide and 0.1g m-phenylenediamine. Then add 20 mL of N, N-dimethylformamide to the polytetrafluoroethylene reactor, and then place it in an oven to react at 250°C for 4 hours. Then the solution was taken out, dialyzed, filtered, and freeze-dried to obtain a platinum-medicated carbon nanodot solid.

Example 3

Weigh 0.1g cisplatin oxide and 1g p-phenylenediamine. Then, 40 mL of dimethyl sulfoxide was added to the polytetrafluoroethylene reactor, and then placed in an oven to react at 180°C for 9 hours. Then the solution was taken out, dialyzed, filtered, and freeze-dried to obtain a platinum-medicated carbon nanodot solid.

Example 4

Weigh 0.5g cisplatin diacid and 1g o-phenylenediamine. Then add 30 mL of N, N-dimethylformamide to the polytetrafluoroethylene reactor, and then place it in an oven to react at 210°C for 8 hours. Then the solution was taken out, dialyzed, filtered, and freeze-dried to obtain a platinum-medicated carbon nanodot solid.

Experimental example one

The structure of the platinum-medicated carbon nanodot compound obtained in Example 1 was subjected to ultraviolet analysis, fluorescence spectrum analysis and XPS (X-ray photoelectron spectroscopy) spectrum analysis.

The test method for characterization of the structure of platinum-medicated carbon nanodots is: using a transmission electron microscope (Tecnai G20 TEM (FEI)), setting the parameters to 200KV

The test method for EDS analysis is: using X-ray energy dispersive spectroscopy (model: Tecnai G20 TEM (FEI)), setting the parameters to 200KV

The test method for XPS analysis is: using an X-ray electron spectrometer (model: ESCALAB 250Xi photoelectron spectrometer), setting Mo as the excitation light source

The test results are as follows:

The transmission electron microscope picture is shown in Figure 2. The size of the carbon nanodots is about 5nm, and the lattice stripe spacing under high magnification is 0.21nm, proving that the platinum carbon nanodots were successfully prepared. The results of EDS (X-ray energy dispersive spectroscopy) and XPS analysis of the elements of the platinum carbon nanodots are shown in Figure 3 and Figure 4 respectively. It can be seen that it has C, N, O, Cl, and Pt signals, indicating that platinum has successfully Participate in the formation of carbon nanodots. The XPS energy spectrum results of Pt show that the electron binding energy of Pt 4f is 75.5eV and 78.8eV, which is a Pt(IV) structural compound (as shown in Figure 5).

Experimental example two

The photoactivation properties of the platinum-medicated carbon nanodot compound obtained in Example 1 were analyzed by XPS spectrum, ¹ HNMR (hydrogen nuclear magnetic resonance spectroscopy) and ESR (electron spin spectroscopy).

The test method for XPS analysis is: the same as Experimental Example 1;

¹ The test method for HNMR analysis is: use a nuclear magnetic resonance spectrometer (model: Bruker Ultra Shield 600 PLUS NMR spectrometer) and set the number of scans to 32-128 times;

The test method for ESR analysis is: using an electron spin resonance spectrometer (model: Bruker EMS Plus), setting the parameters to a magnetic field strength of 3480±175Gauss;

The test results are as follows:

Its XPS energy spectrum before and after illumination shows that the Pt(NH ₃ ) ₂ fitting peak in N 1s and the Pt-O fitting peak in O1s decrease, proving that the platinum drug is released under illumination (Figures 6 and 7). And the XPS spectrum of the sustained-release product collected after illumination shows that the electron binding energy of Pt 4f is between 76.0eV and 72.8eV, which is a Pt(II) structural compound (as shown in Figure 5), proving that the released platinum material is highly divalent. Toxic platinum drugs. At the same time, the XPS spectrum of the platinum-medicated carbon nanodot compound after illumination showed that the COOH fitting peak in O1s increased significantly. This result was also obtained in the ¹ HNMR spectrum (as shown in Figure 8). It is then combined with the electron spin trapping agent DMPO, and what kind of reactive oxygen species are produced by the platinum-medicated carbon nanodots is detected before and after illumination. Electron spin spectroscopy (ESR) results confirmed that no hydroxyl radicals were produced without illumination. Once illuminated, significant hydroxyl radicals were produced (as shown in Figure 9), further demonstrating that platinum-medicated carbon nanodots Compounds have the ability to lower the pH of the surrounding environment.

Experimental example three

The platinum-medicated carbon nanodots of Example 1 are dissolved in one of water, physiological saline, buffer solution, tissue culture fluid or body fluid, wherein the wavelength of the excitation light can be selected from 300nm-1200nm (for example, 589nm in Figure 10 laser); the photodynamic irradiation time can be selected from 0-10 hours (for example, 0.5h in Figure 10); the photodynamic irradiation device can be selected from laser or LED light; the photodynamic power can be selected from 0- Between 5W/cm ² (for example, 0.5W/cm ² in Figure 10). Make it into a solution, and then apply a laser with a wavelength of 400-650nm (for example, 589nm in Figure 10) and a power of 0.1-1.0w/cm ² (for example, 0.5W/cm ² in Figure 10). The platinum medicated carbon The nanodots are reduced under light irradiation to obtain the divalent cisplatin anticancer drug.

Experimental example four

In order to improve the optical properties and endocytosis ability of carbon nanodots, bovine serum albumin was used to composite with the platinum-medicated carbon nanodots of Example 1. The ratio of platinum-medicated carbon nanodots to bovine serum albumin was a mass ratio of 0.5:10. , and conducted transmission electron microscopy observations and UV-visible absorption and fluorescence tests on the composites.

Same experiment example 1;

The UV-visible absorption and fluorescence test methods are: use a UV-visible absorption spectrometer (Model: Shimadzu UV-2600 spectrophotometer), set the 350-800nm range scan, use a fluorescence spectrometer (Model: Edinburgh FS5 spectrophotometer), set the 352-900nm range scan.

Observing the composite nanodots through a transmission electron microscope, it can be seen that after complexing with bovine serum albumin, the size increases from about 5 nm to about 50 nm (as shown in Figure 11). Subsequently, UV-visible absorption and fluorescence tests were carried out on the platinum-medicated carbon nanodots before and after bovine serum albumin complexing. The results are shown in Figure 12. The absorption peak of the UV absorption spectrum did not change significantly, but the fluorescence spectrum curve showed that through the bovine serum albumin Compounding can significantly improve the fluorescence of platinum-medicated carbon nanodots.

Experimental example five

Carry out the drug sustained release test, use a dialysis bag with a molecular weight cutoff of 1000 to load 2 mL of the platinum drug carbon nanodots of Example 1 at a concentration of 0.5 mg/mL, and then place them into 50 mL of different groups using PBS buffer solution as the medium. Including 10mM glutathione (GSH) (i.e., CDs+10mMGSH group) or 589nm laser (5min interval, 0.5W/cm ² ) (i.e., CDs+light group) or control (i.e., CDs group).

The test method for the release rate is: take 2 mL of liquid from outside the dialysis bag at corresponding intervals, and use ICP-MS to measure the Pt content.

The calculation formula of the release rate is: Pt content at the corresponding time point/total Pt content × 100%.

The results are shown in Figure 13. The platinum drug carbon nanodots of Example 1 released the platinum drug rapidly after illumination, and the release rate reached 40% after five times of illumination within 4 hours. However, the platinum drug in the glutathione environment at the same time was The release rate is only about 10%, indicating a good light-controlled release rate. This shows that the release rate is significantly better than reducing media such as glutathione.

Experimental example six

Then the pH value is tested, using a pH meter as the pH value testing method. The platinum-medicated carbon nanodot solutions with different solubilities were illuminated with laser power of 0.1-1.0W/cm ² , irradiation time of 1-30min, and laser wavelength of 380-650nm. For example, as shown in Figure 14, the pH value of the platinum-medicated carbon nanodots of Example 1 decreases under 589nm irradiation. Taking 0.25mg/mL as an example, under 0.5W/ ^cm2 illumination for 10 minutes, the final pH value can decrease. to below 6.5.

Embodiment 7

Subsequently, a cell endocytosis test was performed. The platinum-medicated carbon nanodots of Example 1 and the platinum-medicated carbon nanodots@BSA of Experimental Example 4 were used at the same concentration and at different times to study the intracellular platinum content. The solubility used was 10 μMPt, which was specifically derived from a 6-thioguanine-resistant cell line (the cells were 4T1 breast cancer cells) screened from the 410.4 tumor line without mutagenesis, and the cell concentration was 200,000/well. Through ICP-MS (Inductively Coupled Plasma Mass Spectrometry), the results are shown in Figure 15. As the co-culture time with cells increases, the enrichment of platinum-medicated carbon nanodots@BSA in cells increases significantly, and the enrichment occurs at 12 hours. Enrichment decreases after maximum. Compared with platinum-medicated carbon nanodots, the enrichment capacity of platinum-medicated carbon nanodots after coating with BSA reaches 19 times. Fluorescence microscopy pictures also prove this trend (shown in Figure 16).

Experimental example eight

Subsequently, a light cytotoxicity test was performed. The platinum-medicated carbon nanodots of Example 1 and the platinum-medicated carbon nanodots@BSA of Experimental Example 4 were compared with 4T1 cells (derived from small Mouse breast cancer cells) (cell concentration: 7500 cells/well) were co-cultured. After 6 hours of culture, a 589nm laser was used to irradiate the cells with a power of 0.5W/ ^cm2 for 10min, and then continued to incubate for 48h. In addition, the toxicity test method is: using MTT colorimetric method. The principle is that active cell mitochondria can reduce exogenous MTT into water-insoluble blue-violet crystalline formazan. After dissolving formazan in dimethyl sulfoxide, its light absorption value is measured at a wavelength of 490nm using an enzyme-linked immunoassay detector. Can indirectly reflect the number of viable cells.

The results are as follows: As shown in Figures 17 and 18, the toxicity of the platinum-medicated carbon nanodots in Example 1 increased rapidly after illumination. Compared with pure carbon dots, the concentration of carbon dots wrapped with BSA had a significant inhibitory effect at 10 μM, while Pure carbon dots require 40μM to have obvious inhibitory effect.

Experimental example nine

Subsequently, the death mode and the expression of reactive oxygen species at the cellular level were studied, using cisplatin with a platinum concentration of 10 μM, the platinum-medicated carbon quantum dots of Example 1, and the platinum-medicated carbon quantum dots@BSA of Experimental Example 4 under 589nm laser irradiation. Phosphorylated histone H2AX (p-H2A.X) expression ability of 4T1 breast cancer cells. Agarose gel electrophoresis was used to test the phosphorylated histone H2AX (p-H2A.X) content in 4T1 breast cancer cells. The results are shown in Figure 19. The p-H2A. Drug damage to DNA.

Therefore, the expression of reactive oxygen species was used to study its effect on cell death, and molecular imprinting technology was used for analysis. The method is to obtain different levels of bands based on the different migration rates caused by the size and charge differences of different proteins. The results are shown in Figure 20. Under the action of the ferroptosis inhibitor Fer-1, the ROS produced by platinum-drug carbon quantum dots@BSA on 4T1 cells was partially inhibited, indicating that the activity of platinum-drug carbon quantum dots@BSA produced after illumination Oxygen participates in cellular ferroptosis. As shown in Figure 21, flow cytometry was used to study the death of 4T1 cells after exposure to platinum-medicated carbon quantum dots@BSA. The addition of Fer-1 can inhibit cell death, further indicating that the cells have ferroptosis characteristics.

Embodiment 10

Subsequently, immune cell death (ICD) research was conducted, using cisplatin with a platinum concentration of 10 μM, the platinum-medicated carbon quantum dots of Example 1, and the platinum-medicated carbon quantum dots@BSA of Experimental Example 4 to kill 4T1 breast cancer cells under 589 nm laser irradiation. Immune-related factors: expression of calreticulin (CRT) and high mobility group protein B1 (HMGB1). As shown in Figure 22, the expression of calreticulin on the surface of 4T1 cell membrane and the disappearance of high mobility group protein B1 in the nucleus of medicated carbon quantum dots@BSA under 589nm laser irradiation indicate the induction of immune cell death.

Embodiment 11

Animal experiments show that BABL/c mice with orthotopic 4T1 tumors received a tail vein injection ^when the tumor grew to 80 mm, and 200 μL of cisplatin at a concentration of 2 mg/kg and the platinum drug carbon quantum dots of Example 1 were injected once. , Experimental Example 4 of platinum-medicated carbon quantum dots @BSA, for the illumination group, 589nm laser, 0.5W/cm ² power and 10min irradiation time were used. In addition, the tumor measurement method is: use vernier calipers to measure the length and width of the tumor respectively, and the volume is 1/2 × width ² × length.

The results are shown in Figure 23. The platinum-medicated carbon nanodots@BSA group had a significant inhibitory effect after illumination, indicating that it has a significant light-controlled therapeutic effect. Moreover, the platinum-medicated carbon nanodots@BSA group also had a significant inhibitory effect on distal tumors after illumination (distal tumors are specifically tumors without illumination), indicating a significant immunotherapy effect (Figure 24).

Embodiment 12

Subsequently, all tumors of BABL/c mice with 4T1 tumors were excised after 14 days of treatment, and the mice continued to be observed. The survival curve is shown in Figure 25. The mice treated in the platinum-medicated carbon quantum dot@BSA illumination group of Experimental Example 4 were still alive after 30 days, while all mice in the other control groups died. The mice were then dissected to obtain different lungs. As shown in Figure 26, there was no obvious tumor generation in the lungs of mice treated with platinum-medicated carbon quantum dots@BSA illumination (i.e., Pt-CDs@BSA+laser) group; in addition, compared with the control group, platinum-medicated carbon quantum dots without illumination (Pt-CDs@BSA+laser) -CDs) group and the cisplatin group, the tumor growth in the platinum-medicated carbon quantum dots + light group (i.e., Pt-CDs + laser) was inhibited to a certain extent, but the distal tumors had no obvious inhibitory effect; while other controls groups (i.e., platinum-drug carbon quantum dots@BSA without light (Pt-CDs@BSA) group, platinum-drug carbon quantum dots without light (Pt-CDs) group, and cisplatin group) had a large number of tumors metastasized to the lungs, indicating that platinum drug Carbon quantum dots@BSA light-controlled therapy can produce significant immunity, thereby inhibiting lung metastasis.

In summary, the platinum-medicine carbon nanodots and their protein complexes in the embodiments of the present disclosure have controllable light-responsive release characteristics. Under the condition of illumination, they can be reduced to obtain divalent platinum anti-cancer drugs, which have strong tumor killing properties. It not only reduces the side effects of platinum drugs, but also improves the anti-cancer effect of traditional cisplatin. In addition, compared with traditional single-drug cisplatin, the platinum-drug carbon nanodots in the embodiments of the present disclosure have diverse forms, and their protein complexing can improve the circulation time in the body and enrichment in tumor sites.

The above descriptions are only preferred embodiments of the present disclosure and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this disclosure shall be included in the protection scope of this disclosure.

Industrial applicability

The present disclosure provides a platinum-medicine carbon nanodot and its preparation method, carbon nanodot protein complex and application. The platinum-medicine carbon nanodots and their protein complexes can cause immunogenic death of cancer cells. While killing cancer cells, they can also activate the body's anti-tumor immune response. They can be used as highly efficient photoactive anti-cancer drugs for precision tumor treatment. treatment, so it has excellent practical performance and excellent medical value.

Claims

A platinum drug carbon nanodot, characterized in that it includes a carbon-based core with visible light absorption properties, the carbon-based core is coordinated with a tetravalent platinum element, the carbon-based core has visible light absorption, and the platinum drug Carbon nanodots can be reduced to obtain divalent platinum compounds and hydroxyl radicals under light irradiation conditions. Preferably, the wavelength of light irradiation is 200nm-1200nm.
A method for preparing platinum-medicated carbon nanodots, which is characterized in that it includes: performing a solvothermal reaction between a disubstituted aromatic compound and a tetravalent platinum complex to obtain the platinum-medicated carbon nanodots.
The preparation method of platinum-medicated carbon nanodots according to claim 2, characterized in that the disubstituted aromatic compound is a disubstituted compound of benzene, naphthalene, anthracene or phenanthrene; and/or the tetravalent platinum compound The material is selected from at least one of oxidized cisplatin and diacid cisplatin, and the chemical formula of the oxidized cisplatin is:

Preferably, the disubstituted aromatic compound is an aromatic compound of a diamine.
The preparation method of platinum-medicated carbon nanodots according to claim 2, wherein the disubstituted aromatic compound is a compound represented by formula (I) or formula (II);

In formula (I) and formula (II), R 1 and R 2 are each independently selected from -(CH 2 ) m NH 2 , -O(CH 2 ) n NH 2 , -(CH 2 ) m OH, -O Any one of (CH 2 ) n OH, -(CH 2 ) m NO 2 , -O(CH 2 ) n NO 2 , -(CH 2 ) m COOH, -O(CH 2 ) n COOH; m and n takes 0 to 10 respectively.
The preparation method of platinum-medicated carbon nanodots according to claim 4, characterized in that R 1 and R 2 are each independently selected from -NH 2 , -ONH 2 , -OH, -NO 2 , -ONO 2 , - Any one of COOH and -OCOOH;

And/or, the tetravalent platinum complex is the oxidized cisplatin;

Preferably, the disubstituted aromatic compound is at least one of the following compounds:

More preferably, the disubstituted aromatic compound is
The preparation method of platinum-medicated carbon nanodots according to claim 2, characterized in that the molar ratio of the disubstituted aromatic compound and the tetravalent platinum complex is 1-1000:1-1000, preferably 1 ~100:1~100, more preferably 1~3:1~3.
The preparation method of platinum-medicated carbon nanodots according to claim 6, wherein the reaction solvent is selected from the group consisting of dimethyl sulfoxide, N, N'-dimethylformamide, water, formic acid, ethanol, and ether. , at least one of tetrahydrofuran, folic acid and acetone;

And/or, the ratio of the disubstituted aromatic compound to the solvent is 0.001g/mL～1g/mL.
The preparation method of platinum-medicated carbon nanodots according to claim 6, characterized in that the temperature of the solvothermal reaction is 100-250°C, and the reaction time is 1-10 hours.
A carbon nanodot protein complex, characterized in that it is composed of the platinum-medicated carbon nanodots described in claim 1 or the platinum-medicated carbon nanodots prepared by the preparation method described in any one of claims 2 to 8, and Macromolecule proteins are complexed.
A carbon nanodot protein complex, characterized by comprising the platinum-medicated carbon nanodots described in claim 1 or the platinum-medicated carbon nanodots prepared by the preparation method described in any one of claims 2 to 8, and large molecular proteins;

Optionally, the absorption peak of the ultraviolet absorption spectrum of the carbon nanodot protein complex is in the range of 380-800 nm;

Optionally, the fluorescence emission range of the carbon nanodot protein complex is 500-850nm;

Optionally, the size of the carbon nanodot protein complex ranges from 10 to 500 nm.
The platinum-medicated carbon nanodots according to claim 1 or the platinum-medicated carbon nanodots prepared by the preparation method according to any one of claims 2 to 8 or the carbon nanodot protein composite according to claims 9 or 10 Application of substances in the preparation of anti-tumor drugs.
The platinum-medicated carbon nanodots of claim 1 or the platinum-medicated carbon nanodots prepared by the preparation method of any one of claims 2 to 8 or the carbon nanodot protein complex of claims 9 or 10 are prepared with For use in treating tumors.
A method for treating tumors, characterized in that the method includes: administering the platinum-medicine carbon nanodots of claim 1 or the preparation method of any one of claims 2 to 8 to a subject in need. The platinum-medicine carbon nanodots prepared by the method or the carbon nanodot protein complex according to claim 9 or 10.