Photo-thermal carbon dot compound capable of generating carbon free radicals as well as preparation method and application of photo-thermal carbon dot compound

Abstract

The invention discloses a photo-thermal carbon dot compound capable of generating carbon free radicals as well as a preparation method and application of the photo-thermal carbon dot compound. The photo-thermal carbon dot compound takes a carbon dot as a core, a free radical initiator AIPH is modified through a surface coupling strategy, and photo-thermal and carbon free radical synergistic treatment is achieved. The preparation method comprises the following steps: thermally synthesizing carbon dots (CDs) by using a urea / citric acid solvent, and carrying out EDC / NHS-mediated coupling on AIPH. The compound generates a photothermal effect under 808 nm laser and triggers AIPH to decompose and release carbon free radicals, the drug resistance of tumor heat shock protein is overcome through low-temperature thermal therapy, and the killing capacity on tumor cells is improved through combined treatment with the carbon free radicals. The material has excellent photo-thermal stability, can be expanded to photosensitizer / targeting molecule coupling, is applied to the fields of tumor photo-thermal-thermal power synergistic treatment, microenvironment regulation and control, radiotherapy sensitization and the like, and provides a new strategy for low-toxicity and high-efficiency cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterial preparation, and in particular to a photothermal carbon dot composite capable of generating carbon free radicals, a preparation method thereof, and applications thereof. Background Art

[0002] Cancer, one of the leading causes of death worldwide, poses a serious threat to human life. Traditional cancer treatments, including surgery, radiotherapy, and chemotherapy, often suffer from incomplete treatment, high recurrence and metastasis, severe side effects, and drug resistance. Therefore, there is an urgent need to develop new and effective cancer treatments. Photothermal therapy (PTT), which converts light energy into heat energy to kill cancer cells, is a novel tumor treatment method with the advantages of being non-invasive, having minimal adverse reactions, and being highly targeted. It holds great potential for the development of tumor treatment.

[0003] Currently, carbon dots are widely studied as photothermal agents. However, existing carbon dots face technical bottlenecks in photothermal therapy: high-temperature photothermal therapy can easily damage surrounding normal tissues and may induce upregulation of heat shock proteins to produce heat resistance, thereby reducing efficacy; low-temperature photothermal therapy can make up for the above defects, but its ability to kill tumor cells is limited and its efficacy is slow. Therefore, it is expected to improve the therapeutic effect by combining with other therapies. For example, the carbon dots loaded with the chemotherapy drug doxorubicin disclosed in CN115721731A enhance the killing ability of cancer cells through combined therapy. However, chemotherapy drugs are generally highly toxic and easily damage normal cells. Therefore, the development of a multifunctional, low-toxic carbon dot system with combined therapy is a difficult problem that needs to be solved urgently in this field. Summary of the Invention

[0004] In order to solve the problems of heat resistance and toxicity of chemotherapy drugs raised in the above background technology, the present invention provides a low-toxic carbon dot complex for photothermal-carbon free radical synergistic therapy and its preparation method and application.

[0005] Specifically, the present invention discloses a method for preparing a photothermal carbon dot composite capable of generating carbon free radicals, which comprises the following steps:

[0006] (1) Preparation of surface functionalized carbon dots: amino-containing organic compounds and polycarboxylic acids are mixed in a C1-C3 carboxylic acid solvent, and a solvothermal reaction is performed to generate carbon dots (CDs) with rich surface carboxyl and amino groups.

[0007] (2) Free radical initiator loading: The carboxyl groups on the surface of CDs were activated by EDC / NHS and coupled with the amino groups of the carbon free radical initiator to obtain the final photothermal carbon dot complex.

[0008] For the technical solution described above, it is further preferred that the amino-containing organic compound in step (1) is any one of urea, o-phenylenediamine or ethylenediamine;

[0009] For the technical solutions described above, further preferably, the polybasic carboxylic acid is any one of citric acid, folic acid or ascorbic acid;

[0010] For the technical solutions described above, further preferably, the C1-C3 carboxylic acid solvent is any one of formic acid, acetic acid or propionic acid.

[0011] For the technical solutions described above, further preferably, the solvent thermal reaction of step (1) has an amino-containing organic matter to polybasic carboxylic acid mass ratio of 1:(0.5-1.5).

[0012] For the technical solutions described above, further preferably, the solvent thermal reaction of step (1) has a temperature of 160-200℃ and a reaction time of 3-6 hours.

[0013] For the technical solutions described above, further preferably, the dialysis in step (1) uses a dialysis membrane with a molecular weight cut-off of 500-3000 Da.

[0014] For the technical solutions described above, further preferably, the carbon radical initiator in step (2) is any one of 2,2'-azobis(2-amino propane) dihydrochloride (AIBN) and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] (AIPH).

[0015] For the technical solutions described above, further preferably, the mass ratio of the CDs to the radical initiator is 1:(5-15).

[0016] For the technical solutions described above, further preferably, the coupling reaction in step (2) has a pH value of 6-7.5 and a reaction time of 8-24 hours.

[0017] In another aspect of the present application, a photothermal carbon dot composite prepared by the above method is protected, and the structural formula of the composite is CDs-[radical initiator], wherein when the radical initiator is AIPH, it is denoted as CDs-AIPH. The Zeta potential of the CDs-AIPH is +15 to +25 mV, the photothermal conversion efficiency under 808 nm laser irradiation is ≥35%, and the carbon radical has a sustained release capacity in the range of 45-50℃. The absorbance of ABTS at 740 nm of the CDs-AIPH is increased by 5-10 times of the initial value after 1 hour of stimulation at 45℃. When the concentration of the CDs-AIPH is 200 μg / mL, the cell survival rate is reduced to below 20% under 808 nm laser irradiation, and the CDs-AIPH has a good tumor killing ability.

[0018] In another aspect of the present application, the use of the photothermal carbon dot complex (CDs-[radical initiator]) in the preparation of an anticancer drug or a photothermal treatment material is protected. It synergistically acts through the following mechanisms:

[0019] a) photothermal-carbon radical synergistic killing effect under 808 nm laser excitation;

[0020] b) low-temperature heat-triggered radical release to overcome heat shock protein resistance.

[0021] It couples the radical initiator (such as AIPH) to the surface of the carbon dot through a carboxyl activation strategy, forming a complex that has both photothermal conversion function and controllable radical release ability. It is used for the preparation of photothermal-radical combined antitumor functional preparations, including but not limited to freeze-dried powder, sustained-release injectable dosage forms, etc. More preferably, it is suitable for near-infrared light-responsive tumor ablation materials, and is used for the preparation of antitumor drug intermediates.

[0022] The beneficial effects of the present application are:

[0023] The photothermal carbon dot complex prepared in the present application can produce carbon radicals, and under 808 nm laser irradiation, it can induce the decomposition of the radical initiator to produce carbon radicals, realizing the combined action of photothermal-carbon radicals. This is embodied in:

[0024] ① Through surface coupling strategy, high photothermal conversion efficiency (38.9%) and radical initiator AIPH modification (adjusting Zeta potential to 19.4 mV) are simultaneously realized, overcoming the functional antagonism problem in the modification process;

[0025] ② Low-temperature photothermal (45-50℃) triggers the sustained release of carbon radicals (such as the 7.5-fold increase in absorbance in the ABTS experiment), which can inhibit the heat shock protein resistance;

[0026] ③ The complex can be used as an intelligent treatment platform, and can be coupled with various photosensitizers or targeting molecules to realize material function customization. Its application prospects cover the fields of photothermal ablation, tumor microenvironment regulation, and auxiliary radiosensitization. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the transmission electron microscopy (TEM) image of CDs;

[0028] Figure 2 (a) is the UV absorption and fluorescence spectrum of CDs under 540 nm excitation; (b) is the fluorescence spectrum of CDs under different excitation wavelengths;

[0029] Figure 3 is the FT-IR spectrum of CDs, CDs-AIPH;

[0030] Figure 4 is the zeta potential diagram of CDs and CDs-AIPH aqueous solutions;

[0031] Figure 5 (a) CDs-AIPH at a power density of 1 W cm -2 (a) The temperature rise curves of CDs-AIPH solution at different concentrations under 808 nm laser irradiation; (b) The temperature rise curves of CDs-AIPH solution (200 μg / mL) at different powers; (c) The cyclic temperature rise diagram of CDs-AIPH solution under 808 nm laser irradiation;

[0032] Figure 6 (a) is 200 μg mL -1 (a) is the photothermal effect diagram of CDs-AIPH aqueous solution under 808nm laser irradiation; (b) is the linear relationship diagram of -Ln(θ) and time t.

[0033] Figure 7 This is the UV absorption spectrum of ABTS after adding CDs-AIPH in a 45°C water bath.

[0034] Figure 8 The CDs-AIPH complex is dark-toxic and phototoxic in 4T1 cells. DETAILED DESCRIPTION

[0035] Below in conjunction with the accompanying drawings and Examples, the specific embodiments of the present invention are further described in detail. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise stated, the materials such as chemical reagents used in the present invention are obtained from commercial sources and are processed and used in accordance with standard operating procedures. All experimental steps were carried out under conventional laboratory conditions to ensure the repeatability and reliability of the technical solution.

[0036] Example 1

[0037] 1. Preparation of CDs-AIPH

[0038] (1) Preparation of surface functionalized carbon dots

[0039] 2 g of urea and 1 g of citric acid were dissolved in 10 mL of formic acid by ultrasonic dissolution, and then transferred to a reactor for solvothermal reaction at 180°C for 4 h. After the reaction was completed and cooled to room temperature, ethanol was added to the reaction solution, and the precipitate was collected by centrifugation. The precipitate was dissolved in deionized water, and the supernatant was collected by centrifugation. CDs were obtained by dialyzing and freeze-dried for later use.

[0040] (2) Free radical initiator loading

[0041] Dissolve CDs (10 mg) in deionized water, add EDC (40 mg) and NHS (50 mg), and stir at room temperature to activate the surface carboxyl groups of the CDs. Add AIPH (100 mg) to the activated CDs solution and stir until the reaction is complete. Dialyze to obtain CDs-AIPH, which is then lyophilized for later use.

[0042] Figure 1 This is a transmission electron microscope (TEM) image of the prepared carbon dots (CDs). The carbon dots are spherical and about 5 nm in size.

[0043] Figure 2 (a) is the UV absorption spectrum of CDs and the fluorescence spectrum under 540 nm excitation. CDs has a broad absorption band in the near-infrared region, indicating that it has the ability of photoacoustic imaging and near-infrared light-triggered PTT. Figure 2 (b) is the fluorescence spectra of CDs at different excitation wavelengths, indicating that CDs are excitation independent.

[0044] Figure 3 The FT-IR spectra of CDs and CDs-AIPH are shown below. -1 and 3190cm -1 The characteristic peak at 1629cm corresponds to the stretching vibration of OH and NH; -1 The characteristic peak at 3350cm corresponds to the stretching vibration of C=O, which proves that the surface of CDs contains a large number of carboxyl and amino groups. -1 The characteristic OH peak at 37° was significantly weakened, proving that AIPH was successfully coupled.

[0045] The Zeta potential of CDs and CDs-AIPH aqueous solutions was tested. Figure 4 The zeta potential of CDs is -31.8 mV, indicating that there are a large number of carboxyl groups on the surface of CDs. The zeta potential of AIPH is 4.3 mV, and the zeta potential of CDs-AIPH is 19.4 mV, indicating that AIPH is successfully coupled to the surface of CDs.

[0046] Based on the strong near-infrared absorption ability of carbon dots, we further tested the photothermal performance of CDs-AIPH under 808nm laser irradiation. Figure 5 As shown in (a), the prepared CDs-AIPH was prepared into concentration gradients of 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL, respectively, and the power density was 1 W cm -2The temperature of the solution increased with the increase of concentration. The temperature of the 200μg / mL CDs-AIPH solution rose to 52.3℃, while the temperature of the aqueous solution remained almost unchanged. Afterwards, the photothermal performance of CDs-AIPH was tested at different laser power densities. Figure 5 As shown in (b), a CDs-AIPH solution with a concentration of 200 μg / mL was exposed to 808 nm laser light with a power density of 0.6 W cm -2 , 0.8W cm -2 、1Wcm -2 、1.5W cm -2 With the increase of laser power, the temperature rises, indicating that the temperature rise of CDs-AIPH has significant power dependence. Figure 5 (c) is the photothermal stability diagram of CDs-AIPH. Even after 5 consecutive switching laser irradiations, CDs-AIPH still exhibits a stable photothermal effect, indicating that CDs-AIPH has good photothermal stability.

[0047] In order to measure the photothermal conversion efficiency of CDs-AIPH, the samples were irradiated with 808 nm laser for 10 min and then naturally cooled for 10 min. The photothermal conversion efficiency (η) was calculated according to the following formula.

[0048]

[0049] Where h is the heat transfer coefficient and S is the surface area of ​​the container. max，mix and ΔT max，H2O are the temperature changes of CDs-AIPH and H2O at the maximum stable temperature, respectively. I is the laser power, and A is the absorbance of CDs-AIPH in aqueous solution at a wavelength of 808 nm. s is the sample system time constant, m D and c D is the solvent mass and heat capacity, θ is the dimensionless driving force temperature, defined as the relationship between ΔT and ΔT max,mix ratio.

[0050] like Figure 6 (a) is 200 μg mL -1 Photothermal effect diagram of CDs-AIPH aqueous solution under 808nm laser irradiation; Figure 6 (b) is the linear relationship between -Ln(θ) and time t. Calculated, 200μg mL -1 The photothermal conversion efficiency of CDs-AIPH aqueous solution under 808nm laser irradiation can reach 38.9%.

[0051] Figure 7This is the UV absorption spectrum of ABTS after adding CDs-AIPH in a 45°C water bath. ABTS's absorption at 740 nm increases after interacting with carbon radicals. When the CDs-AIPH solution containing ABTS is treated in a 45°C water bath, the ABTS absorption at 740 nm gradually increases, demonstrating that the decomposition of AIPH coupled to the carbon dots generates carbon radicals.

[0052] Figure 8 The dark toxicity and phototoxicity of CDs-AIPH complex in 4T1 cells (mouse breast cancer cells) were tested by MTT assay. 5 A 4T1 cell suspension at 1 μg / mL was seeded into each well of a 96-well plate. 100 μL of DMEM medium containing 10% FBS was added to each well and incubated for 12 hours. The medium was then removed using a pipette. Different concentrations of CDs-AIPH (0, 6.25, 12.5, 25, 50, 100, and 200 μg / mL) in DMEM medium were added to each well. After a further 10-hour incubation, the phototoxicity test group was irradiated with an 808 nm (1 W) laser for 10 minutes. After a further 12-hour incubation, the medium was removed again, and 100 μL of DMEM medium containing MTT (0.5 mg / mL) was added to each well of the 96-well plate. After a 4-hour incubation, the solution in each well was removed, leaving purple formazan crystals, and 100 μL of DMSO solvent was added to each well. Transfer the 96-well plate to a microplate reader and shake for 30 seconds. Measure the absorbance of the formazan solution at 490 nm. Calculate cell viability according to the following formula.

[0053] Cell viability = (OD CD -ODK CD ) / (OD control -ODK control )

[0054] Among them, OD CD Refers to the absorbance value after treatment with different concentrations of CDs-AIPH, ODK CD Refers to the absorbance value of the blank well containing only DMSO, OD control represents the absorbance value of the control group without CDs-AIPH treatment, ODK control Refers to the absorbance value of DMSO, and each experiment was repeated 4 times.

[0055] As the concentration of CDs-AIPH increased, the cell survival rate remained above 85% even at 200 μg / mL, indicating low dark toxicity of CDs-AIPH. However, in the light-treated experimental group, at a concentration of 200 μg / mL, the cell survival rate dropped below 20%, demonstrating significant phototoxicity under 808 nm laser irradiation.

[0056] It should be understood that the above embodiments are intended only to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection thereof. Persons skilled in the art may make various modifications and variations to the above embodiments without departing from the spirit and basic principles of the present invention, but such modifications and variations shall still fall within the scope of protection of the claims of the present invention and their equivalents. The scope of protection of the present invention shall be based on the appended claims and shall not be limited to the details of the above embodiments.

Claims

1. A method for preparing a photothermal carbon dot composite capable of generating carbon free radicals, characterized in that: The following steps are involved: (1) An amino-containing organic compound and a polycarboxylic acid are mixed in a C1-C3 carboxylic acid solvent, and a solvothermal reaction is performed to generate carbon dots (CDs) with rich surface carboxyl and amino groups. (2) The carboxyl groups on the surface of CDs were activated by EDC / NHS and coupled with the amino groups of the carbon free radical initiator to obtain a photothermal carbon dot complex.

2. The preparation method according to claim 1, characterized in that In step (1), the amino-containing organic compound is any one of urea, o-phenylenediamine or ethylenediamine; the polycarboxylic acid is any one of citric acid, folic acid or ascorbic acid; and the C1-C3 carboxylic acid solvent is any one of formic acid, acetic acid or propionic acid.

3. The preparation method according to claim 1, characterized in that The mass ratio of the amino-containing organic matter to the polycarboxylic acid in the solvent thermal reaction of step (1) is 1:(0.5-1.5).

4. The preparation method according to claim 1, characterized in that The solvent thermal reaction temperature of step (1) is 160-200° C., and the reaction time is 3-6 hours.

5. The preparation method according to claim 1, characterized in that The carbon free radical initiator in step (2) is AIBN or AIPH.

6. The preparation method according to claim 1, characterized in that The mass ratio of the CDs to the carbon radical initiator is 1:(5-15).

7. The preparation method according to claim 1, characterized in that The pH value of the coupling reaction in step (2) is 6-7.5, and the reaction time is 8-24 hours.

8. The photothermal carbon dot composite prepared by the method of claim 1, wherein: The zeta potential of the photothermal carbon dot complex in step (2) is +15 to +25 mV, the photothermal conversion efficiency under 808 nm laser irradiation is ≥35%, and it has the ability to continuously release carbon free radicals in the range of 45-50° C. After stimulation at 45° C. for 1 hour, the absorbance increase of ABTS at 740 nm reaches 5-10 times the initial value.

9. The photothermal carbon dot composite according to claim 8, characterized in that: When the concentration of the photothermal carbon dot complex is 200 μg / mL, the cell survival rate is reduced to below 20% under 808 nm laser irradiation.

10. Use of the photothermal carbon dot composite according to claim 8 in the preparation of anticancer drugs or photothermal therapeutic materials.

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