CN113694197A

CN113694197A - Photothermal/photodynamic synergistic tumor phototherapy reagent and preparation method and application thereof

Info

Publication number: CN113694197A
Application number: CN202111269075.6A
Authority: CN
Inventors: 党闪闪; 张斌; 聂国辉; 吴汉伟; 李华伟; 韩景宏; 王雨洁; 曾俊清
Original assignee: Shenzhen Second Peoples Hospital
Current assignee: Shenzhen Second Peoples Hospital
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2021-11-26
Anticipated expiration: 2041-10-29
Also published as: CN113694197B

Abstract

The invention discloses a photothermal/photodynamic synergistic tumor phototherapy reagent, which comprises a metal cluster, a photosensitizer and an MXene nanosheet material, wherein the metal cluster and the photosensitizer are loaded on the MXene nanosheet material to form an MXene nanosheet composite material; meanwhile, a preparation method of the photo-thermal/photodynamic synergistic tumor phototherapy reagent and application of the photo-thermal/photodynamic synergistic tumor phototherapy reagent are also disclosed. The invention utilizes MXene material to exert photothermal effect and photodynamic activity, and utilizes metal clusters to decompose excessive hydrogen peroxide or water in tumor cells into oxygen to realize continuous oxygen supply for photosensitizer and generate active oxygen species, thereby further enhancing photodynamic therapy.

Description

Photothermal/photodynamic synergistic tumor phototherapy reagent and preparation method and application thereof

Technical Field

The invention belongs to the technical field of materials and biological medicines, relates to an anti-tumor phototherapy reagent and a preparation method and application thereof, and particularly relates to a photothermal/photodynamic synergistic tumor phototherapy reagent and a preparation method and application thereof.

Background

In recent years, the incidence and fatality rate of malignant tumors are gradually increased, which seriously threatens the life and health of human beings, and cancer and the treatment thereof are long-standing problems. The traditional tumor treatment method has certain defects, such as obvious side effect of chemotherapy, limited inhibition of tumor growth caused by drug resistance and the like. Therefore, the development of new methods for tumor-targeted therapy with high efficacy and less side effects has become one of the most important issues in the medical field. With the continuous development of photosensitive materials and nanotechnology, phototherapy provides a minimally invasive and efficient treatment approach for tumor treatment.

Phototherapy is an emerging therapeutic approach, is a tumor treatment mode which is initiated by light, is noninvasive and efficient, and mainly comprises two therapies of photo-thermal therapy and photodynamic therapy. Photothermal therapy is based on the generation of heat when light energy is absorbed by tumor tissue, such as blood and cholesterol, which can cause irreversible thermal damage to the tumor tissue and thus ablation of the tumor. However, photothermal therapy may cause problems such as damage to surrounding tissues due to local overheating. Photodynamic therapy is a photochemical reaction utilizing a photosensitizer, wherein the photosensitizer capable of being selectively absorbed by tumor tissues generates singlet oxygen, hydroxyl free radicals and other active oxygen species under the irradiation of laser with specific wavelength, and can trigger pathological reaction in tumor cells to cause cell damage or apoptosis, thereby achieving a therapeutic effect. In the process of photodynamic therapy, the photosensitizer can be rapidly distributed in tissues after entering a human body and is mostly discharged out of the body after several days; the generated active oxygen species have small diffusion distance and short half-life period in physiological environment; the photosensitizer can induce the phototoxic action only when reaching a certain concentration and being irradiated by sufficient light; therefore, the photodynamic therapy has the advantages of minimal invasion, low toxicity and low drug resistance, and mainly adopts the photothermal therapy as the auxiliary and the photodynamic therapy as the way of realizing efficient tumor treatment. Although great progress has been made in recent years, the clinical application research is limited due to the problems of cell hypoxia, photosensitizer aggregation and the like.

In the last decade, compared with common optical materials, the two-dimensional MXene nanosheet has a superior photo-thermal conversion efficiency due to the unique nanostructure and physicochemical properties thereof, and has been widely applied to biomedical nanomaterials. Since 2011 proposed by academicians in billows of our country, the development of novel carrier-supported metal monatomic and cluster materials has been developed as the leading edge of research in the field of heterogeneous catalysis. The two-dimensional material-supported metal material which is emerging in the field of photoelectricity in recent years has a wide research prospect in optical treatment.

Disclosure of Invention

In order to solve the above problems, an object of the present invention is to provide a photothermal/photodynamic synergistic tumor phototherapy reagent based on palladium-loaded metal clusters, which employs a metal material based on MXene loading, and is applied to photothermal/photodynamic synergistic tumor therapy to achieve an efficient tumor phototherapy effect, and at the same time, a preparation method of the photothermal/photodynamic synergistic tumor phototherapy reagent and an application of the photothermal/photodynamic synergistic tumor phototherapy reagent are provided.

In order to achieve the purpose, the invention adopts the following technical scheme:

the tumor phototherapy reagent with photo-thermal/photodynamic synergism comprises a metal cluster, a photosensitizer and an MXene nanosheet material, wherein the metal cluster and the photosensitizer are loaded on the MXene nanosheet material to form an MXene nanosheet composite material; the loading amount of the metal clusters is 2-6 wt% of the mass of the carrier, and the loading amount of the photosensitizer is 1-6 wt% of the mass of the carrier.

Further, MXene is Ti₃C₂T_x、Ti₂CT_x、Nb₂C or Ta₄C₃T_xNanosheets of which T_xRepresenting terminal group on the surface, including-OH, -O or-F, and the size of the nano-sheet is 50-300 nm; the photosensitizer is 5,10,15, 20-tetra (4-aminobenzene) -21H, 23H-porphyrin, chlorin E6 or indocyanine green; the metal cluster includes 20 to 100 atoms.

A preparation method of a tumor phototherapy reagent with photo-thermal/photodynamic synergy comprises the following steps:

s1, dispersing MXene powder in an ice water bath through a probe in an ultrasonic manner to prepare a suspension with uniformly dispersed MXene;

s2, dissolving a photosensitizer in a first solvent by ultrasonic in an ice water bath to prepare a photosensitizer solution;

s3, dissolving the metal salt in a second solvent to prepare a metal salt solution;

s4, dropwise adding the metal salt solution obtained in the step S3 into deionized water, uniformly stirring, adding the first solution, uniformly stirring, adding the sodium hydroxide water solution, uniformly stirring, adding the sodium borohydride water solution, and continuously uniformly stirring to obtain a mixed solution;

s5, dialyzing and purifying the mixed solution obtained in the step S4 in ultrapure water by using a dialysis bag, and freezing and storing the obtained metal cluster solution at-20 ℃ for later use;

s6, adding the photosensitizer solution obtained in the step S2 and the cluster solution obtained in the step S5 into the MXene suspension obtained in the step S1, and performing common ultrasound in an ice water bath to obtain uniformly dispersed suspension;

s7, placing the suspension obtained in the step S6 in a refrigerator at 4 ℃ to be shaken up, and then carrying out centrifugal treatment to obtain a tumor phototherapy reagent; the tumor phototherapy agent was re-ultrasonically dispersed in deionized water in an ice water bath and stored at 4 ℃ until use.

Further, in the step S1, the MXene nanosheets are Ti₃C₂T_x、Ti₂CT_x、Nb₂C or Ta₄C₃T_xThe concentration of the nano-sheet and MXene suspension is 0.5-4 mg/mL.

Further, in the above step S2, the first solvent is absolute ethanol or dimethyl sulfoxide.

Further, in the step S3, the second solvent is one or more of deionized water, absolute ethyl alcohol, and acetone.

Further, in the step S3, the metal salt is palladium nitrate, palladium chloride, chloroplatinic acid or chloroauric acid, and the concentration of the metal salt solution is 0.01mol/L to 0.05 mol/L.

Further, in the step S4, the first solution is an aqueous solution of glutathione or an aqueous solution of 3-mercaptopropionic acid, and the concentration is 0.003mol/L to 0.01 mol/L; the concentration of the sodium hydroxide aqueous solution is 0.5-1.5 mol/L; the concentration of the sodium borohydride aqueous solution is 0.05 mol/L-0.2 mol/L.

Further, in the step S4, the first solution is added and stirred for 5-20 min; adding a sodium hydroxide aqueous solution and stirring for 5-20 min; and finally adding a sodium borohydride aqueous solution and continuously stirring for 3-24 hours.

Further, in the above step S5, the cut-off molecular weight of the dialysis bag is 7kDa to 15 kDa.

Further, in the step S6, the normal ultrasound time in the ice-water bath is 0.5 to 4 hours.

The invention also aims to provide application of the photothermal/photodynamic synergistic tumor phototherapy reagent in preparation of a photothermal/photodynamic synergistic tumor phototherapy reagent.

Further, in the above technical solution, the tumor comprises SW480 human colon cancer cells.

Due to the adoption of the technical scheme, the invention has the following advantages:

the tumor phototherapy reagent with photo-thermal/photodynamic synergy uses the two-dimensional nano-sheet MXene as a carrier, the ultrathin MXene has larger specific surface area and rich terminal groups, a large number of anchoring sites are provided for photosensitizer, chemotherapeutic drugs and the like, and the composite material can be obtained by simple stirring and mixing; meanwhile, MXene shows strong absorption characteristic and photothermal conversion performance in a near-infrared region, and is cooperated with a photosensitizer, so that the application of the two-dimensional MXene material in the biological field is enriched.

The tumor phototherapy reagent with photo-thermal/photodynamic cooperation utilizes MXene materials to exert photo-thermal effect and photodynamic activity; the toxicity to cells is very low under the condition of no illumination, and the self-quenching effect of free photosensitizer is overcome; compared with metal nano materials, the application of the metal clusters obviously reduces the metal toxicity, improves the metal utilization rate and reduces the metal waste; meanwhile, the photosensitizer is loaded on the surface of the MXene nanosheet, so that the self-quenching effect of the photosensitizer is overcome, and the service life of the photosensitizer is prolonged. The tumor phototherapy reagent can decompose excessive hydrogen peroxide or water in tumor cells into oxygen by utilizing the metal clusters, so that continuous oxygen supply and active oxygen species generation are realized for the photosensitizer, the photodynamic therapy is further enhanced, the photodynamic phototherapy reagent is applied to photo-thermal/photodynamic synergistic therapy of tumors to realize efficient tumor phototherapy effect, and the metal clusters have good market prospect.

Drawings

Fig. 1 is a TEM picture of the metal cluster-photosensitizer-MXene of the present invention;

FIG. 2 is a graph of photothermal curves of pure MXene, photosensitizer-MXene, Pd-photosensitizer-MXene;

FIG. 3a shows the results of addition of different reagents H in the dark₂O₂O of solution₂A concentration variation graph;

FIG. 3b shows the results of addition of different reagents under light conditions H₂O₂O of solution₂A concentration variation graph;

FIG. 4 is a bar graph of the toxicity evaluation of the tumor phototherapy agents of the present invention against SW480 cells, MHCC97L cells, CAKI cells and A549 cells in the absence of light;

FIG. 5 is a bar graph of toxicity evaluation of pure MXene, photosensitizer-MXene, Pd-photosensitizer-MXene in light on SW480 cells;

fig. 6 is a picture of HE stained sections of major organs 14 days after mouse injection of samples, scale bar =50 um;

FIG. 7a is a graph of relative tumor volume versus time in mice in the absence of light;

FIG. 7b is a graph of mouse tumor relative volume as a function of time under light conditions.

Detailed Description

The present invention will be further described in detail with reference to the following examples; however, the following examples are merely illustrative, and the present invention is not limited to these examples.

Example 1

first, 23 mg of Pd (NO)₃)₂Adding the solution into 10 mL of deionized water, and fully stirring the solution to prepare a metal palladium salt solution with the metal ion concentration of 0.01 mol/L; adding 2.61 mu L of 3-mercaptopropionic acid into 9997.39 mu L of deionized water, and fully stirring to prepare a 3-mercaptopropionic acid solution with the concentration of 0.003 mol/L; adding 0.6 g NaOH into 10 ml deionized water, fully and uniformly stirring to prepare a sodium hydroxide solution with the concentration of 1.5 mol/L; 4.75 mg NaBH₄Adding the mixture into 2.5ml of deionized water, and fully shaking the mixture to prepare 0.05mol/L aqueous solution of sodium borohydride;

then, 0.25 ml of the metal palladium salt solution is measured and added into 2.35 ml of deionized water, the mixture is fully and evenly stirred, 2ml of the 3-mercaptopropionic acid solution is added and stirred for 5min, then 0.3 ml of the NaOH solution is added and stirred for 20 min, and finally 0.1 ml of the NaBH is added₄Stirring the solution for 24 hours; dialyzing and purifying the obtained mixed solution in ultrapure water for 3 times and 3 hours each time by using a dialysis bag with the molecular weight cutoff of 7kDa, and freezing and storing the obtained palladium cluster solution at-20 ℃ for later use;

then, 1mg of 5,10,15, 20-tetra (4-aminobenzene) -21H, 23H-porphyrin is added into 10 ml of absolute ethyl alcohol, and the mixture is subjected to ultrasonic treatment for 30 min to prepare a porphyrin solution with the concentration of 0.1 mg/ml; 0.5 mg of Nb with a plate diameter size of 50 nm₂Adding the C powder into 1mL of deionized water, and preparing into uniformly dispersed Nb through probe ultrasonic dispersion₂C, suspension liquid; 0.2 mL of the palladium cluster solution and 0.05 mL of the porphyrin solution were measured and added to the Nb₂C suspensionIn the preparation method, ultrasonic treatment is carried out for 0.5 h in an ice-water bath, the mixture is placed in a refrigerator at 4 ℃ for overnight vibration and shaking up, and then the mixture is centrifugally treated to obtain a tumor phototherapy reagent, namely: 2% Pd-1% photosensitizer-Nb₂C; the tumor phototherapy reagent is ultrasonically dispersed in deionized water again and stored at 4 ℃ for standby.

Example 2

first, 46 mg of Pd (NO)₃)₂Adding the solution into 10 mL of deionized water, and fully stirring the solution to prepare a metal palladium salt solution with the metal ion concentration of 0.02 mol/L; adding 12.3 mg of glutathione into 10 mL of deionized water, and fully stirring to prepare a glutathione aqueous solution with the concentration of 0.004 mol/L; adding 0.52 g NaOH into 10 mL deionized water, and fully and uniformly stirring to prepare a sodium hydroxide solution with the concentration of 1.3 mol/L; 7.6 mg NaBH₄Adding the mixture into 2.5mL of deionized water, and fully shaking the mixture to prepare a solution with the concentration of 0.08 mol/L;

then, 0.25 ml of the metal palladium salt solution is measured and added into 2.35 ml of deionized water, the mixture is fully and evenly stirred, 2ml of glutathione solution is added and stirred for 7 min, then 0.3 ml of the NaOH solution is added and stirred for 18 min, and finally 0.1 ml of the NaBH is added₄Stirring the solution for 12 hours; dialyzing and purifying the obtained mixed solution in ultrapure water for 3 times and 3 hours each time by using a dialysis bag with the molecular weight cutoff of 7kDa, and freezing and storing the obtained palladium cluster solution at-20 ℃ for later use;

then, 1mg of 5,10,15, 20-tetra (4-aminobenzene) -21H, 23H-porphyrin is added into a mixed solution of 5mL of absolute ethyl alcohol and 5mL of dimethyl sulfoxide, and the mixture is subjected to ultrasonic treatment for 30 min to prepare a porphyrin solution with the concentration of 0.1 mg/mL; 2 mg of Ta having a plate size of 100 nm and surface terminal groups of mainly-OH₄C₃T_xAdding into 2mL deionized water, and preparing into uniformly dispersed Ta by ultrasonic dispersion₄C₃T_xA suspension; 0.6ml of the palladium cluster solution and 0.6ml of the porphyrin solution were measured and added to the Ti₃C₂T_xSuspending in water, ultrasonic treating for 1 hr in ice water bath, and shaking in refrigerator at 4 deg.CHomogenization followed by centrifugation yielded the tumor phototherapy reagents, namely: 3% Pd-3% photosensitizer-Ta₄C₃T_x(ii) a The tumor phototherapy reagent is ultrasonically dispersed in deionized water again and stored at 4 ℃ for standby.

Example 3

first, 155 mg of H₂Cl₆Adding Pt into 10 mL of deionized water, fully stirring and preparing a metal platinum salt solution with the metal ion concentration of 0.03 mol/L; adding 4.35 mu L of 3-mercaptopropionic acid into 9995.65 mu L of deionized water, and fully stirring to prepare a 3-mercaptopropionic acid solution with the concentration of 0.005 mol/L; adding 0.4 g NaOH into 10 mL deionized water, and fully and uniformly stirring to prepare a sodium hydroxide solution with the concentration of 1.0 mol/L; 9.5 mg NaBH₄Adding the mixture into 2.5mL of deionized water, and fully shaking the mixture to prepare a solution with the concentration of 0.1 mol/L;

then, 0.25 mL of the platinum salt solution was weighed and added to 2.35 mL of deionized water, followed by stirring, 2mL of the 3-mercaptopropionic acid solution was added and stirred for 10min, 0.3 mL of the NaOH solution was added and stirred for 16 min, and finally 0.1 mL of the NaBH solution was added₄The solution is stirred for 3 hours; dialyzing and purifying the obtained mixed solution in ultrapure water for 3 times and 3 hours each time by using a dialysis bag with the molecular weight cutoff of 10 kDa, and freezing and storing the obtained platinum cluster solution at-20 ℃ for later use;

then, 1mg of indocyanine green is added into 10 mL of dimethyl sulfoxide, and the mixture is subjected to ultrasonic treatment for 30 min to prepare a porphyrin solution with the concentration of 0.1 mg/mL; 1mg of Ti having a sheet diameter of 200 nm and surface terminal groups of mainly-F₃C₂T_xAdding the Ti into 1mL of deionized water, and preparing the Ti with uniform dispersion through ultrasonic dispersion₃C₂T_xA suspension; 0.137 mL of the platinum cluster solution and 0.6mL of the porphyrin solution were measured and added to the Ti₃C₂T_xIn the suspension, carrying out ultrasonic treatment in an ice-water bath for 2 h, placing the suspension in a refrigerator at 4 ℃ for shaking up, and then carrying out centrifugal treatment to obtain a tumor phototherapy reagent, namely: 4% Pt-6% photosensitizer-Ti₃C₂T_x(ii) a The tumor phototherapy agent was re-ultrasonically dispersed in deionized water and stored at 4 ℃ until use.

Example 4

first, 259 mg of H₂Cl₆Adding Pt into 10 mL of deionized water, fully stirring and preparing a metal platinum salt solution with the metal ion concentration of 0.05 mol/L; adding 21.5 mg of glutathione into 10 mL of deionized water, and fully stirring to prepare a glutathione aqueous solution with the concentration of 0.007 mol/L; adding 0.32 g NaOH into 10 mL deionized water, and fully and uniformly stirring to prepare a sodium hydroxide solution with the concentration of 0.8 mol/L; 13.3 mg NaBH₄Adding the mixture into 2.5mL of deionized water, and fully shaking the mixture to prepare a solution with the concentration of 0.14 mol/L;

then, 0.25 mL of the platinum salt solution was weighed and added to 2.35 mL of deionized water, followed by stirring, 2mL of the glutathione solution was added and stirred for 13 min, 0.3 mL of the NaOH solution was added and stirred for 12 min, and finally 0.1 mL of the NaBH solution was added₄The solution is stirred for 10 hours; dialyzing and purifying the obtained mixed solution in ultrapure water for 3 times and 3 hours each time by using a dialysis bag with the molecular weight cutoff of 9 kDa, and freezing and storing the obtained palladium cluster solution at-20 ℃ for later use;

then, 1mg of chlorin E6 is added into 10 mL of absolute ethyl alcohol, and the mixture is subjected to ultrasonic treatment for 30 min to prepare a porphyrin solution with the concentration of 0.1 mg/mL; 2 mg of Ti having a sheet diameter of 300 nm and surface terminal groups of mainly-F₂CT_xAdding the Ti into 1mL of deionized water, and preparing the Ti with uniform dispersion through ultrasonic dispersion₂CT_xA suspension; 0.246 mL of the platinum cluster solution and 0.2 mL of the porphyrin solution were measured and added to the Ti₂CT_xIn the suspension, carrying out ultrasonic treatment in an ice-water bath for 2.5 h, placing the suspension in a refrigerator at 4 ℃ for shaking up, and then carrying out centrifugal treatment to obtain a tumor phototherapy reagent, namely: 6% Pt-1% photosensitizer-Ti₂CT_x(ii) a The tumor phototherapy agent was re-ultrasonically dispersed in deionized water and stored at 4 ℃ until use.

Example 5

first, 35 mg of PdCl₂Adding the solution into a mixed solution of 5mL of absolute ethyl alcohol and 5mL of acetone, and fully stirring the solution to prepare a metal palladium salt solution with the metal ion concentration of 0.02 mol/L; adding 6.96 mu L of 3-mercaptopropionic acid into 9993.04 mu L of deionized water, and fully stirring to prepare a 3-mercaptopropionic acid solution with the concentration of 0.008 mol/L; adding 0.28 g of NaOH into 10 mL of deionized water, and fully and uniformly stirring to prepare a sodium hydroxide solution with the concentration of 0.7 mol/L; 15.2 mg NaBH₄Adding the mixture into 2.5mL of deionized water, and fully shaking the mixture to prepare a solution with the concentration of 0.16 mol/L;

then, 0.25 mL of the above metal palladium salt solution was weighed and added to 2.35 mL of deionized water, stirred well, 2mL of the above 3-mercaptopropionic acid solution was added and stirred for 15 min, then 0.3 mL of the above NaOH solution was added and stirred for 9 min, and finally 0.1 mL of the above NaBH₄The solution is stirred for 18 hours; dialyzing and purifying the obtained solution in ultrapure water for 3 times and 3 hours each time by using a dialysis bag with the molecular weight cutoff of 12 kDa, and freezing and storing the obtained palladium cluster solution at-20 ℃ for later use;

then, adding 1mg of indocyanine green into a mixed solution of 4 mL of anhydrous ethanol and 6mL of dimethyl sulfoxide, and carrying out ultrasonic treatment for 30 min to prepare a porphyrin solution with the concentration of 0.1 mg/mL; 1mg of Nb with a sheet diameter of 150 nm₂C is added into 1mL deionized water and prepared into uniformly dispersed Nb by ultrasonic dispersion₂C, suspension liquid; 0.2 mL of the palladium cluster solution and 0.2 mL of the porphyrin solution were measured and added to the Nb₂C, performing ultrasonic treatment on the suspension in an ice-water bath for 3 hours, placing the suspension in a refrigerator at 4 ℃ for shaking up, and then performing centrifugal treatment to obtain a tumor phototherapy reagent, namely: 2% Pd-2% photosensitizer-Nb₂C; the tumor phototherapy reagent is ultrasonically dispersed in deionized water again and stored at 4 ℃ for standby.

Example 6

first of all, the first step is to,102 mg of AuCl₄H is added into 10 mL of deionized water, and the mixture is fully stirred to prepare a metal gold salt solution with the metal ion concentration of 0.03 mol/L; adding 27.7 mg of glutathione into 10 mL of deionized water, and fully stirring to prepare a glutathione aqueous solution with the concentration of 0.009 mol/L; adding 0.24 g of NaOH into 10 mL of deionized water, and fully and uniformly stirring to prepare a sodium hydroxide solution with the concentration of 0.6 mol/L; 17.1mg NaBH₄Adding the mixture into 2.5mL of deionized water, and fully shaking the mixture to prepare a solution with the concentration of 0.18 mol/L;

then, 0.25 mL of the above metal gold salt solution was weighed and added to 2.35 mL of deionized water, stirred well, 2mL of the above glutathione aqueous solution was added and stirred for 18 min, then 0.3 mL of the above NaOH solution was added and stirred for 7 min, and finally 0.1 mL of the above NaBH was added₄The solution is stirred for 8 hours; dialyzing and purifying the obtained mixed solution in ultrapure water for 3 times and 3 hours each time by using a dialysis bag with the molecular weight cutoff of 7kDa, and freezing and storing the obtained gold cluster solution at-20 ℃ for later use;

then, 1mg of chlorin E6 is added into a mixed solution of 5mL of absolute ethyl alcohol and 5mL of dimethyl sulfoxide, and the mixed solution is subjected to ultrasonic treatment for 30 min to prepare a porphyrin solution with the concentration of 0.1 mg/mL; 2 mg of Ta having a sheet diameter of 200 nm and surface terminal groups of mainly-OH₄C₃T_xAdding into 0.5 mL deionized water, and ultrasonically preparing into uniformly dispersed Ta₄C₃T_xA suspension; 0.272 mL of the gold cluster solution and 0.8 mL of the porphyrin solution were measured and added to the Ti₂CT_xIn the suspension, carrying out ultrasonic treatment in an ice-water bath for 3.5h, placing the suspension in a refrigerator at 4 ℃ for shaking up, and then carrying out centrifugal treatment to obtain a tumor phototherapy reagent, namely: 4% Au-4% photosensitizer-Ta₄C₃T_x(ii) a The tumor phototherapy agent was re-ultrasonically dispersed in deionized water and stored at 4 ℃ until use.

Example 7

first, 170 mg of AuCl was added₄H is added into a mixed solution of 5mL of deionized water and 5mL of absolute ethyl alcohol,fully stirring to prepare a metal gold salt solution with the metal ion concentration of 0.05 mol/L; adding 8.7 mu L of 3-mercaptopropionic acid into 9991.3 mu L of deionized water, and fully stirring to prepare a 0.01 mol/L3-mercaptopropionic acid solution; adding 0.2 g NaOH into 10 mL deionized water, and fully and uniformly stirring to prepare a sodium hydroxide solution with the concentration of 0.5 mol/L; 19 mg of NaBH₄Adding the mixture into 2.5mL of deionized water, and fully shaking the mixture to prepare a solution with the concentration of 0.2 mol/L;

then, 0.25 mL of the above metal gold salt solution was weighed and added to 2.35 mL of deionized water, and stirred well for 10min, 2mL of the above 3-mercaptopropionic acid solution was added and stirred for 10min, then 0.3 mL of the above NaOH solution was added and stirred for 10min, and finally 0.1 mL of the above NaBH solution was added₄The solution is stirred for 6 hours; dialyzing and purifying the obtained mixed solution in ultrapure water for 3 times and 3 hours each time by using a dialysis bag with the molecular weight cutoff of 7kDa, and freezing and storing the obtained gold cluster solution at-20 ℃ for later use;

then, 1mg of indocyanine green is added into 10 mL of dimethyl sulfoxide, and the mixture is subjected to ultrasonic treatment for 30 min to prepare a porphyrin solution with the concentration of 0.1 mg/mL; 1mg of Ti having a sheet diameter of 300 nm and surface terminal groups of mainly-O₃C₂T_xAdding the Ti powder into 0.5 mL of deionized water, and preparing the Ti powder into uniformly dispersed Ti powder by ultrasonic dispersion₃C₂T_xA suspension; 0.102 mL of the gold cluster solution and 0.6mL of the porphyrin solution were measured and added to the Ti₃C₂T_xIn the suspension, carrying out ultrasonic treatment for 4h in an ice-water bath, placing the suspension in a refrigerator at 4 ℃ for shaking up, and then carrying out centrifugal treatment to obtain a tumor phototherapy reagent, namely: 6% Au-6% photosensitizer-Ti₃C₂T_x(ii) a The tumor phototherapy agent was re-ultrasonically dispersed in deionized water and stored at 4 ℃ until use.

Application example 1

As shown in fig. 1, a TEM image of the photothermal/photodynamic synergistic tumor phototherapy agent of the present invention obtained in the above example 3 is shown in fig. 1, and it can be obtained from fig. 1: the MXene is still in a thin two-dimensional nanosheet structure after loading the metal clusters and the photosensitizer, and the diameter of the nanosheet structure is 300 nm.

The invention relates to photo-thermal/photodynamic synergetic tumor phototherapy reagent detection

(1) Photo-thermal properties of the material

Near-infrared light irradiation is adopted to test the photo-thermal performance of MXene, the MXene modified by photosensitizer (namely photosensitizer-MXene in figure 2), the photosensitizer and the MXene material modified by metal cluster (namely metal cluster-photosensitizer-Mxene in figure 2 and the tumor phototherapy reagent of the invention)

1.0 mL of an aqueous solution in which 50 ppm of a single material was dispersed was placed in a 1.5mL centrifuge tube, fixed on a base, and irradiated with a laser beam at 808nm (power density of light irradiation: 1W/cm)²) The temperature rise of the sample was recorded with a thermal imager for 10min, and the results are shown in FIG. 2.

From the analysis of the results in fig. 2, it can be seen that the modification of the photosensitizer and the metal cluster has no significant effect on the photothermal effect of MXene.

(2) Experiment for catalyzing hydrogen peroxide or water to generate oxygen by material

The catalytic performance of the sample for catalyzing hydrogen peroxide to generate oxygen is tested by means of a dissolved oxygen meter (JPB-707A)

Measuring 150. mu.L of 1 mg/L sample and 150. mu.L hydrogen peroxide (30%) into 2.7 mL of deionized water, and monitoring the oxygen content in the solution without light (in dark), the results are shown in FIG. 3 a; monitoring the oxygen content in the solution under the condition of illumination, wherein the illumination condition is as follows: 808nm, 1W/cm²The results are shown in FIG. 3 b.

From the analysis of the results in fig. 3a and 3b, the metal cluster-photosensitizer-MXene of the present invention shows superior catalytic performance to that of pure MXene and photosensitizer-MXene under both non-illumination and illumination conditions, and the catalytic performance is enhanced by near infrared 808nm illumination. This demonstrates that the modified metal cluster-photosensitizer-MXene is able to catalyze the decomposition of excess hydrogen peroxide in cells to oxygen to achieve a continuous oxygen supply to the photosensitizer and the generation of reactive oxygen species to further enhance photodynamic therapy.

(3) Cytotoxicity test

The research on the toxicity of the photo-thermal/photodynamic synergetic tumor phototherapy reagent on a series of tumor cells is carried out by means of a CCK-8 kit

First, SW480 cells, MHCC97L cells, CAKI cells and A549 cells were seeded in a 96-well plate (1.2X 10 cells per well)⁴Individual cells) were cultured in DMEM medium at 37 ℃ under 5% carbon dioxide for 24 hours, then, monatomic material solutions (0, 12.5, 25, 50, 100, 200, 400 μ g/mL) of different concentrations were added to continue culturing the cells for 24 hours, and finally, the viability of the cells was measured using a standard CCK-8 kit, and the results are shown in fig. 4.

As can be seen from the analysis in fig. 4, after the tumor phototherapy agent (metal cluster-photosensitizer-MXene) of the present invention is cultured in a medium containing the tumor phototherapy agent (metal cluster-photosensitizer-MXene) for 24 hours under non-illumination conditions, the survival rate of SW480 cells, MHCC97L cells, CAKI cells, and a549 cells is still maintained at 85% or more at a material concentration of 400 μ g/mL, which indicates that the material has very low toxicity to cells and has little effect on cell viability, thereby proving that the tumor phototherapy agent of the present invention has good biocompatibility.

(4) Cytotoxicity test

In vitro phototherapy experiment

SW480 cells are inoculated in a 96-well plate for culturing for 24h, the culture medium respectively selects fresh culture media of MXene, photosensitizer-MXene and metal cluster-photosensitizer-MXene with different concentrations, and the MXene content in materials with different concentrations in the experimental process is the same (0, 12.5, 25, 50 and 100 mu g/mL); after culturing for 4h, the power is 1W/cm²Irradiating the cells with 808nm near infrared light for 5min, continuously culturing for 24h, and detecting the activity of the cells by using a standard CCK-8 kit. The results are shown in FIG. 5.

As can be seen from the analysis of fig. 5, compared with pure MXene and photosensitizer-MXene, the cytotoxicity of the phototherapy agent for tumor of the present invention, i.e., metal cluster-photosensitizer-MXene, is significantly improved under the near-infrared light irradiation condition.

(5) Biological safety test

The toxicity of MXene series materials in organisms is researched by using BALB/c female mice of Zhejiang Weitonglihua laboratory animal technology Limited company

Mice were randomly divided into 4 groups of 5 mice each and injected with 4 different reagents as follows: 1) Saline PBS (control); 2) pure MXene; 3) MXene as photosensitizer; 4) metal cluster-photosensitizer-MXene (tumor phototherapy agent of the present invention); injecting 100 mu L (4mg/mL) once, measuring the body weight of the mouse every other day, killing the mouse at the 15 th day, taking blood of the mouse, separating serum, and measuring the levels of inflammatory factors such as TNF alpha, IL-1 beta, IL-6 and the like in the blood of the mouse through an ELISA experiment to evaluate the immune toxicity; meanwhile, the heart, liver, spleen, lung and kidney of the mouse are taken, and whether MXene series materials damage main organs of the mouse is evaluated through an immunohistochemical experiment. The results are shown in FIG. 6.

From the analysis of fig. 6, pure MXene, photosensitizer-MXene and metal cluster-photosensitizer-MXene had no significant effect on mouse heart, liver, spleen, lung and kidney tissues.

(6) Phototherapy experiment for in vivo tumor

In vivo tumor phototherapy experiments using BALB/c female mice

The formula is adopted: tumor volume =1/2 x length x width squared tumor models were calculated. When the tumor grows to about 50 mm³When the mice are divided into 8 groups at random, 6 mice in each group are injected with 4 different reagents, namely 1) group and 2) group, and normal saline PBS; 3) group and 4) group, pure MXene; 5) group 6) group, photosensitizer-MXene; 7) group 8) metal cluster-photosensitizer-MXene; 3) group-8) group was injected with 100 μ L (500 μ g/mL) of reagent once every other day via tail vein, 1) group and 2) group were injected with equal amount of physiological saline once every other day via tail vein; wherein the group 1), the group 3), the group 5) and the group 7) are control groups, and the group 2), the group 4), the group 6) and the group 8) are experimental groups. After 4 hours of reagent injection each time, the utilization power is 0.75W/cm²The 808nm near infrared light irradiates the tumors of the mice in the groups 2) 4) 6) 8) 3 min, and the tumor temperature in the light irradiation process is recorded by an infrared camera. On day 14, mice were sacrificed and treated tumors were obtained by dissection and weighed to assess the final tumor treatment effect; simultaneously fixing tumor tissue, slicing, staining with Ki67 and CD31, and comparing data under different treatment parameters, such as tumor proliferation ability and blood vessel number changeAnd (4) transforming. Meanwhile, the heart, liver, spleen, lung and kidney of the mouse are taken, and whether MXene series materials damage the main organs of the mouse is evaluated through an immunity group experiment. The results are shown in FIGS. 7a and 7 b.

From FIG. 7a, tumor growth was not inhibited in four groups of mice in the absence of light. Referring to fig. 7b, photo-thermal/photodynamic synergistic tumor therapy using the tumor phototherapy agent (metal cluster-photosensitizer-MXene) of the present invention can significantly inhibit tumor growth in mice under illumination conditions compared with normal saline PBS, pure MXene, and photosensitizer-MXene.

Claims

1. A tumor phototherapy reagent with photo-thermal/photodynamic synergy is characterized in that: the photosensitive material comprises a metal cluster, a photosensitizer and an MXene nanosheet material, wherein the metal cluster and the photosensitizer are loaded on the MXene nanosheet material to form an MXene nanosheet composite material; the loading amount of the metal clusters is 2-6 wt% of the mass of the carrier, and the loading amount of the photosensitizer is 1-6 wt% of the mass of the carrier.

2. The photothermal/photodynamic synergistic oncological phototherapy agent according to claim 1, characterized in that: wherein MXene is Ti₃C₂T_x、Ti₂CT_x、Nb₂C or Ta₄C₃T_xNanosheets of which T_xRepresenting terminal group on the surface, including-OH, -O or-F, and the size of the nano-sheet is 50-300 nm; the photosensitizer is 5,10,15, 20-tetra (4-aminobenzene) -21H, 23H-porphyrin, chlorin E6 or indocyanine green; the metal cluster includes 20 to 100 atoms.

3. A method for preparing the photothermal/photodynamic synergistic phototherapy agent for tumor as claimed in claim 1 or 2, characterized in that: which comprises the following steps:

4. The method of preparing a photothermal/photodynamic synergistic phototherapy reagent for tumor as claimed in claim 3, wherein: in step S1, MXene nanosheets are Ti₃C₂T_x、Ti₂CT_x、Nb₂C or Ta₄C₃T_xThe concentration of the nano-sheet and MXene suspension is 0.5-4 mg/mL.

5. The method of preparing a photothermal/photodynamic synergistic phototherapy reagent for tumor as claimed in claim 3, wherein: in step S2, the first solvent is absolute ethanol or dimethyl sulfoxide.

6. The method of preparing a photothermal/photodynamic synergistic phototherapy reagent for tumor as claimed in claim 3, wherein: in step S3, the second solvent is one or more of deionized water, absolute ethyl alcohol, and acetone.

7. The method of preparing a photothermal/photodynamic synergistic phototherapy reagent for tumor as claimed in claim 3, wherein: in step S3, the metal salt is palladium nitrate, palladium chloride, chloroplatinic acid or chloroauric acid, and the concentration of the metal salt solution is 0.01 mol/L-0.05 mol/L.

8. The method of preparing a photothermal/photodynamic synergistic phototherapy reagent for tumor as claimed in claim 3, wherein: in step S4, the first solution is glutathione water solution or 3-mercaptopropionic acid water solution, and the concentration is 0.003 mol/L-0.01 mol/L; the concentration of the sodium hydroxide aqueous solution is 0.5-1.5 mol/L; the concentration of the sodium borohydride aqueous solution is 0.05 mol/L-0.2 mol/L.

9. Use of the photothermal/photodynamic synergistic tumor phototherapy agent of claim 1 or 2 in an agent for the preparation of photothermal/photodynamic synergistic phototherapy of a tumor.

10. Use according to claim 9, characterized in that: the tumor comprises SW480 human colon cancer cells.