Gold nano radiotherapy sensitizer for surface-enhanced Raman spectroscopy and preparation method and application of gold nano radiotherapy sensitizer

Abstract

The invention relates to a gold nano radiotherapy sensitizer for surface enhanced Raman spectroscopy and a preparation method and application thereof. Specifically, the invention provides a composite material with manganese dioxide wrapped by gold nanoparticles, which has improved surface enhanced Raman spectrum signals, improved X-ray deposition efficiency and improved tumor killing effect in radiotherapy. And a nucleic acid aptamer for targeting tumors can be carried, so that the targeting property and the safety are improved, and wide application prospects are realized in the fields of radiotherapy sensitization, tumor diagnosis and the like. In addition, the invention further provides a method for preparing the gold nano composite material, dopamine-grafted polyethylene glycol, gold nano particles, potassium permanganate and the like in different proportions are used as raw materials, and the gold nano composite materials with different shapes and sizes can be prepared and used for different purposes. The preparation method is simple, easy to operate and beneficial to industrial production and application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a gold nanometer radiotherapy sensitizer based on surface enhanced Raman spectroscopy, and a preparation method and application thereof. BACKGROUND

[0002] A radiotherapy sensitizer refers to a compound or drug capable of enhancing the effect of radiotherapy. Radiotherapy sensitizers can increase the sensitivity of tumor cells to radiation in various ways, thereby improving the efficacy of radiotherapy, such as increasing the degree of damage to DNA in tumor cells, inhibiting the repair capacity of cells to radiation damage, etc. Radiotherapy sensitizers are widely studied and applied in clinical practice, and are expected to improve the killing effect of radiotherapy on tumors, reduce the treatment dose, reduce the toxic and side effects, and thus improve the effect of tumor treatment. Multifunctional nano radiotherapy sensitizers provide new opportunities for enhancing the radiosensitivity of tumor cells and improving the effect of radiotherapy.

[0003] The signal intensity of ordinary Raman spectroscopy is very weak. When a molecule is close to or on the surface of an optical laser nanostructure, the Raman signal of the molecule will be greatly enhanced, which is called surface enhanced Raman spectroscopy (SERS). SERS uses the rough surface of noble metals (gold, silver, copper, etc.) to enhance the Raman scattering signal of the adsorbed active molecules by millions to hundreds of millions of times. SERS technology can detect and identify tumor-related markers, cell surface molecules and metabolic products, thereby achieving early diagnosis, treatment monitoring and risk assessment of tumors.

[0004] The application of SERS of gold nanoparticles has the characteristics of high sensitivity, high selectivity, non-destructiveness and multifunctionality, and has wide application prospects in the fields of chemistry, materials science, etc. In addition, gold nanoparticles are a kind of nanomaterials with controllable size, good biocompatibility and high radiation absorption efficiency, which can enhance the killing effect of radiotherapy on tumor cells through various mechanisms. However, the application effect of gold nanomaterials in the medical field, especially in the field of tumor diagnosis and treatment, still needs to be improved.

[0005] Therefore, there is an urgent need in the art to develop gold nanocomposites with enhanced Raman signals. SUMMARY

[0006] The purpose of the present application is to provide gold nanocomposites with enhanced Raman signals and a preparation method and application thereof.

[0007] In a first aspect of the present application, a gold nanocomposite is provided, the composite comprising: (i) a manganese dioxide core; and (ii) gold nanoparticles distributed on the surface;

[0008] wherein the gold nanocomposite has one or more properties selected from the group consisting of:

[0009] (1) improved surface-enhanced Raman spectroscopy signal level;

[0010] (2) improved X-ray deposition efficiency;

[0011] (3) improved level of ROS free radicals in tumor cells;

[0012] (4) improved tumor killing effect in radiotherapy.

[0013] In another preferred embodiment, the gold nanocomposite has a particle size of 100-600 nm, preferably 100-200 nm.

[0014] In another preferred embodiment, the gold nanocomposite has a molar ratio of Au:Mn of (1.1-1.3):1.

[0015] In another preferred embodiment, the gold nanocomposite has a molar ratio of Au:Mn of 1.2:1.

[0016] In another preferred embodiment, the gold nanoparticles are polyethylene glycol modified gold nanoparticles.

[0017] In another preferred embodiment, the nanocomposite further comprises: (iii) a targeting component on the surface of the gold nanoparticles.

[0018] In another preferred embodiment, the targeting component comprises an aptamer, hyaluronic acid, folic acid, RGD peptide.

[0019] In another preferred embodiment, the targeting component is a tumor-targeting aptamer.

[0020] In another preferred embodiment, the aptamer has a sequence as shown in SEQ ID NO: 1.

[0021] In another preferred embodiment, the aptamer is a thiol-modified aptamer.

[0022] In a second aspect of the present application, a method for preparing the gold nanocomposite as described in the first aspect of the present application is provided, comprising the steps of:

[0023] (a) grafting eight-arm polyethylene glycol with dopamine to react with gold nanoparticles to obtain dopamine-polyethylene glycol modified gold nanoparticles;

[0024] (b) subjecting the dopamine-polyethylene glycol modified gold nanoparticles obtained in step (a) to redox reaction with potassium permanganate and bovine serum albumin to obtain the gold nanocomposite as described in the first aspect of the present application.

[0025] In another preferred embodiment, step (a) comprises:

[0026] (a1) adding eight-arm polyethylene glycol (8-arm-PEG) and dopamine hydrochloride into anhydrous polar solvent to react to generate dopamine grafted eight-arm polyethylene glycol (8-arm-PEG-DA);

[0027] (a2) mixing and reacting dopamine grafted eight-arm polyethylene glycol (8-arm-PEG-DA) in step (a1) with gold nanoparticles to obtain dopamine-polyethylene glycol modified gold nanoparticles (Au-PEG-DA).

[0028] In another preferred embodiment, the polar solvent in step (a1) is selected from the group consisting of DMF, DMSO, tetrahydrofuran, methanol; preferably DMF.

[0029] In another preferred embodiment, the molar ratio of eight-arm polyethylene glycol to dopamine hydrochloride in step (a1) is 1:4.

[0030] In another preferred embodiment, the average molecular weight of 8-arm-PEG in step (a2) is 10-40 kDa, preferably 10-20 kDa, and more preferably 10 kDa.

[0031] In another preferred embodiment, the molar ratio of dopamine grafted eight-arm polyethylene glycol (8-arm-PEG-DA) to gold nanoparticles in step (a2) is (10-5):(2-1).

[0032] In another preferred embodiment, the molar ratio of 8-arm-PEG-DA to gold nanoparticles in step (a2) is 10:1.

[0033] In another preferred embodiment, the pH of the reaction system in step (a2) is 5.5-7.4, preferably 7-7.4.

[0034] In another preferred embodiment, the reaction temperature in step (a2) is 37°C±2°C.

[0035] In another preferred embodiment, the reaction time in step (a2) is 24h±2h.

[0036] In another preferred embodiment, the molar ratio of dopamine-polyethylene glycol modified gold nanoparticles to bovine serum albumin in step (b) is (4-2):1.

[0037] In another preferred embodiment, the molar ratio of dopamine-polyethylene glycol modified gold nanoparticles to bovine serum albumin in step (b) is 4:1.

[0038] In another preferred embodiment, the mass fraction of potassium permanganate in the reaction system in step (b) is 2-10%, preferably 2-4%.

[0039] In another preferred embodiment, the reaction temperature of step (b) is 35-45 °C, preferably 40 °C.

[0040] In another preferred embodiment, the reaction time of step (b) is 2±0.5 h, preferably 2 h.

[0041] In another preferred embodiment, the method further comprises a step of:

[0042] (c) modifying a targeting moiety to the surface of the product of step (b).

[0043] In another preferred embodiment, the targeting moiety comprises a nucleic acid aptamer, hyaluronic acid, folic acid, RGD peptide.

[0044] In another preferred embodiment, the targeting moiety is a tumor-targeting nucleic acid aptamer.

[0045] In another preferred embodiment, the sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1.

[0046] In another preferred embodiment, the nucleic acid aptamer is a thiol-modified nucleic acid aptamer.

[0047] In a third aspect of the present application, there is provided a use of the gold nanocomposite as described in the first aspect of the present application for preparing an agent, the agent being used for one or more purposes selected from the group consisting of:

[0048] (1) tumor radiotherapy sensitization;

[0049] (2) tumor diagnosis;

[0050] (3) tumor imaging;

[0051] (4) identification and localization of tumor cells.

[0052] In another preferred embodiment, the tumor diagnosis is early tumor diagnosis.

[0053] In another preferred embodiment, the agent is administered to a subject by injection.

[0054] In another preferred embodiment, the subject is a human or mammal suffering from a tumor.

[0055] In a fourth aspect of the present application, there is provided a tumor radiotherapy sensitization agent comprising the gold nanocomposite as described in the first aspect of the present application.

[0056] In another preferred embodiment, the tumor radiotherapy sensitization agent further comprises a pharmaceutically acceptable carrier.

[0057] In another preferred embodiment, the tumor radiotherapy sensitization agent is administered to a subject by injection.

[0058] In another preferred embodiment, the subject is a human or mammal having a tumor.

[0059] In another preferred embodiment, the radiotherapy comprises X-ray radiotherapy.

[0060] In a fifth aspect of the present application, there is provided a diagnostic reagent comprising the gold nanocomposite as described in the first aspect of the present application.

[0061] In another preferred embodiment, the diagnostic reagent further comprises a diagnostically acceptable carrier.

[0062] In another preferred embodiment, the diagnostic reagent is a tumor diagnostic reagent.

[0063] In another preferred embodiment, the diagnostic reagent can be used for tumor imaging, identification and localization of tumor cells.

[0064] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 The transmission electron microscope (TEM) photos of the nanomaterials prepared in Comparative Example 1 and Example 6 of the present application are shown. Among them, A is the Au-PEG particles of Comparative Example 1, with a size of about 15 nm; B is the Mn@AuNP prepared in Example 6, in which the gold nanoparticles are uniformly distributed on the surface of the manganese dioxide carrier, showing a nanoflower shape, with a size of about 120 nm.

[0066] Figure 2 The hydrated particle size column chart of the nanomaterials prepared in Examples 1 to 6 of the present application is shown.

[0067] Figure 3 The Raman spectrum of the nanomaterials prepared in Comparative Example 1 and Example 6 of the present application in tumor cells is shown.

[0068] Figure 4 The level of ROS free radicals generated by the nanomaterials prepared in Comparative Example 1 and Example 6 of the present application in tumor cells is shown.

[0069] Figure 5 The effects of the nanomaterials prepared in Comparative Example 1 and Example 6 of the present application and the blank control group on the activity of tumor cells are shown.

[0070] Figure 6The in vivo anti-tumor activity of the nanomaterials prepared in Comparative Example 1 and Example 6 of the present application is shown. A is a blank control group with only PBS; B is PBS+X-ray; C is the nanomaterial of Comparative Example 1 +X-ray; D is the nanomaterial of Example 6 +X-ray. DETAILED DESCRIPTION

[0071] Through extensive and in-depth research, the present inventors provide a gold nanoparticle-wrapped manganese dioxide composite material (i.e., "the gold nanoparticle composite material of the present application"), which has an improved surface-enhanced Raman spectroscopy signal, an improved X-ray deposition efficiency, can increase the level of ROS free radicals in tumor cells, thereby improving the tumor killing effect in radiotherapy; and can also carry a tumor-targeting aptamer, and has better tumor targeting and safety. The gold nanoparticle composite material of the present application can be applied to radiotherapy sensitization and tumor imaging.

[0072] In addition, the present application also provides a method for preparing the gold nanoparticle composite material of the present application, which uses dopamine-grafted polyethylene glycol, gold nanoparticles and potassium permanganate, etc. as raw materials in different proportions to prepare gold nanoparticle composite materials with different morphologies and sizes for different uses. The preparation method of the present application is simple and easy to operate, and is conducive to industrialized production and application. On this basis, the present application is completed.

[0073] The gold nanoparticle composite material of the present application

[0074] As used herein, the terms "the gold nanoparticle composite material of the present application", "the gold nanoparticle-wrapped manganese dioxide of the present application", "the Mn@AuNP of the present application" and "the nanoradiotherapy sensitizer of the present application" can be used interchangeably, and all refer to the gold-manganese dioxide composite material of the present application.

[0075] The gold nanoparticle composite material of the present application includes a manganese dioxide nanocore, and gold nanoparticles distributed on the surface thereof. The particle size of the gold nanoparticle composite material of the present application is 100-600 nm, and is preferably 100-200 nm. A typical transmission electron microscope photograph of the gold nanoparticle composite material of the present application is shown in FIG. 1B, which has a particle size of about 125 nm and a nanoflower shape, and the gold nanoparticles are uniformly distributed on the surface of the manganese dioxide core. Figure 1

[0076] In an embodiment of the present application, the gold nanoparticle composite material can further include a targeting component on the surface of the gold nanoparticles for targeting tumors. The targeting component includes an aptamer, hyaluronic acid, folic acid, and RGD peptide. Preferably, the targeting component is a tumor-targeting aptamer.

[0077] ​Aptamer is a single-stranded DNA or RNA oligonucleotide, which has low molecular weight, is easy to synthesize and modify, and has significant target specificity, affinity, good stability, low toxicity and low immunogenicity, can quickly penetrate tissues, and can be applied to the diagnosis and targeted treatment of various cancers.

[0078] In the present application, the thiol group-containing aptamer is reacted with the gold nanoparticles in the composite material, so that the surface of the gold nanoparticle composite material of the present application is provided with the aptamer, so as to have better tumor targeting and improve the effect of the gold nanoparticle composite material of the present application.

[0079] In an embodiment of the present application, the sequence of the aptamer is as follows: TTGGTGGTGGTGGTTGTGGTGGTGGTGG (SEQ ID NO: 1).

[0080] Preparation method

[0081] The present application also provides a preparation method of the gold nanoparticle composite material of the present application. The method uses dopamine-grafted eight-arm polyethylene glycol (8-arm-PEG-DA) and gold nanoparticles as main raw materials, first modifies the gold nanoparticles with dopamine-grafted polyethylene glycol to obtain dopamine-polyethylene glycol modified gold nanoparticles (Au-PEG-DA); then prepares manganese dioxide nanoparticles coated with gold nanoparticles by redox reaction of Au-PEG-DA, bovine serum albumin and potassium permanganate; and finally, optionally modifies tumor-targeting aptamer on the surface of the manganese dioxide nanoparticles coated with gold nanoparticles.

[0082] Specifically, the preparation method of the gold nanoparticle composite material of the present application comprises the following steps:

[0083] (a) reacting dopamine-grafted eight-arm polyethylene glycol (8-arm-PEG-DA) with gold nanoparticles to obtain dopamine-polyethylene glycol modified gold nanoparticles (Au-PEG-DA);

[0084] (b) subjecting Au-PEG-DA obtained in step (a) to redox reaction with potassium permanganate (KMnO4) and bovine serum albumin (BSA) to obtain the gold nanoparticle composite material of the present application.

[0085] In an embodiment of the present application, the structure of the 8-arm-PEG-DA is shown as formula I:

[0086]

[0087] In one embodiment of the present application, step (a) specifically comprises: adding 8-arm-PEG and dopamine hydrochloride into anhydrous polar solvent to react to form 8-arm-PEG-DA; mixing 8-arm-PEG-DA with gold nanoparticles to react to obtain Au-PEG-DA.

[0088] Preferably, the polar solvent is selected from the group consisting of DMF, DMSO, tetrahydrofuran, methanol; preferably DMF.

[0089] Preferably, the molar ratio of 8-arm-PEG to dopamine hydrochloride is 1:4.

[0090] Preferably, the average molecular weight of 8-arm-PEG is 10-40 kDa, preferably 10-20 kDa, more preferably 10 kDa.

[0091] Preferably, the molar ratio of 8-arm-PEG-DA to gold nanoparticles is (10-5):(2-1), more preferably 10:1.

[0092] Preferably, the pH of the reaction of step (a) is 5.5-7.4, more preferably 7-7.4.

[0093] Preferably, the reaction time of step (a) is 24 h±2 h.

[0094] Preferably, the molar ratio of Au-PEG-DA to BSA is (4-2):1, more preferably 4:1.

[0095] Preferably, the content of KMnO4 added is 2-10%, more preferably 2-4%.

[0096] Preferably, the reaction temperature of step (b) is 35-45°C, more preferably 40°C.

[0097] Preferably, the reaction time of step (b) is 2±0.5 h, more preferably 2 h.

[0098] In one embodiment of the present application, the method further comprises the step of:

[0099] (c) modifying a tumor-targeting nucleic acid aptamer to the surface of the product of step (b). The nucleic acid aptamer is a thiol-modified nucleic acid aptamer. Preferably, the sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1.

[0100] Application

[0101] The gold nanocomposite of the present application can be used as a radiotherapy sensitizer. Firstly, gold nanoparticles can accumulate inside and outside tumor cells, increasing the reaction cross section of tissues or cells with X-rays, improving the ability to produce reactive oxygen species (ROS) during radiotherapy, and thus improving the damage to tumor cells. Secondly, the surface plasmon resonance effect of gold nanoparticles can increase the local electron dose, thereby increasing the radioactivity damage to cells. In addition, gold nanoparticles can also enhance the radiosensitivity of cells by generating oxygen radicals or interfering with the repair ability of cells during the repair process of radiation-induced DNA double-strand breaks. The gold nanocomposite of the present application is rich in more gold nanoparticles, further improving the radiotherapy sensitization effect, and the tumor cell killing effect reaches more than 60%.

[0102] The gold nanocomposite of the present application has improved surface-enhanced Raman signal (SERS) level, strong SERS signal, good repeatability, high sensitivity, high selectivity, non-destructive and multi-functionality, and can be used as a SERS probe for tumor recognition, positioning, imaging, diagnosis, etc.

[0103] The main advantages of the present application include:

[0104] 1. The gold nanocomposite of the present application has more gold nanoparticles on a single nanocarrier, thus has more significant radiotherapy sensitization effect, can improve the level of ROS free radicals in tumor cells, and at the same time improve the deposition efficiency of X-rays.

[0105] 2. The gold nanocomposite of the present application has improved surface-enhanced Raman signal level, can be used for tumor diagnosis, and has high sensitivity, high selectivity and non-destructive.

[0106] 3. The gold nanocomposite of the present application as a radiotherapy sensitizer can be used for single irradiation to reduce the irradiation dose.

[0107] 4. The gold nanocomposite of the present application has enhanced stability and good biocompatibility, and has improved safety and feasibility of clinical application.

[0108] 5. The method for preparing the gold nanocomposite in the present application is simple, easy to operate and control, and convenient for industrialized production.

[0109] 6. The method for preparing the gold nanocomposite in the present application can adjust the size and morphology of the nanoparticles by changing the ratio of gold nanoparticles, dopamine grafted eight-arm polyethylene glycol and bovine serum albumin, and the pH value and reaction time of the reaction system, which can be used in various application fields such as radiotherapy sensitizers, tumor marker detection, tumor cell recognition and positioning, etc.

[0110] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate but not limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and fractions are weight percentages and weight fractions.

[0111] Materials and Methods

[0112] The gold nanoparticles in the following examples were prepared by the following method:

[0113] 10 mL of sodium citrate solution (2 mg / mL) was added to the reactor, heated to boiling under magnetic stirring, 2 mL of chloroauric acid solution (1 mg / mL) was added, the solution turned light pink after heating was stopped, the solution was cooled to 90-95℃, the same amount of chloroauric acid solution was added and reacted for 30-40 min, and then slowly cooled to room temperature at room temperature, and the gold nanoparticles were collected by centrifugation (12000 rpm, 15 min).

[0114] Example 1: Method 1 for preparing gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0115] (1) Preparation of gold nanoparticle sensitizer (Au-PEG-DA)

[0116] Under nitrogen protection, 8-arm-PEG (weight average molecular weight 40 kDa) powder and dopamine hydrochloride were added to anhydrous DMF solvent at a molar ratio of 1:4, and stirred at 40℃ for 2h. After the reaction was completed, the reaction solution was poured into pre-iced anhydrous petroleum ether, and the product was filtered after precipitation. After washing with pure water, it was freeze-dried to obtain SG-PEG-DA (i.e. 8-arm-PEG-DA). SG-PEG-DA (weight average molecular weight 40 kDa) and gold nanoparticles were dissolved in PBS (pH = 7.4) solution at a molar ratio of 10:1, and stirred at 37℃ for 24h. After the reaction was completed, the product was collected by centrifugation and freeze-dried to obtain Au-PEG-DA.

[0117] (2) Preparation of gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0118] Au-PEG-DA (weight average molecular weight 40 kDa) and BSA were dissolved in pure water solution at a molar ratio of 2:1, then 2% KMnO4 was added by mass ratio, stirred at 40 °C for 2 h, and the product was collected by centrifugation after the reaction was completed. Finally, the thiol-modified AS1411 nucleic acid aptamer was modified on the nano-radiotherapy sensitizer, and Mn@AuNP was obtained after freeze-drying.

[0119] Example 2: Method 2 for preparing gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0120] (1) Preparation of gold nano-sensitizer (Au-PEG-DA)

[0121] Under nitrogen protection, 8-arm-PEG (weight average molecular weight 20 kDa) powder and dopamine hydrochloride were added to anhydrous DMF solvent at a molar ratio of 1:4, stirred at 40 °C for 2 h, and then the reaction solution was poured into pre-iced anhydrous petroleum ether. After the product was precipitated, it was filtered, washed with pure water, and freeze-dried to obtain SG-PEG-DA. SG-PEG-DA (weight average molecular weight 20 kDa) and gold nanoparticles were dissolved in PBS (pH = 7.4) solution at a molar ratio of 10:1, stirred at 37 °C for 24 h, and the product was collected by centrifugation after the reaction was completed. Freeze-drying obtained Au-PEG-DA.

[0122] (2) Preparation of gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0123] Au-PEG-DA (weight average molecular weight 20 kDa) and BSA were dissolved in pure water solution at a molar ratio of 2:1, then 2% KMnO4 was added by mass ratio, stirred at 40 °C for 2 h, and the product was collected by centrifugation after the reaction was completed. Finally, the thiol-modified AS1411 nucleic acid aptamer was modified on the nano-radiotherapy sensitizer, and Mn@AuNP was obtained after freeze-drying.

[0124] Example 3: Method 3 for preparing gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0125] (1) Preparation of gold nano-sensitizer (Au-PEG-DA)

[0126] Under the protection of nitrogen, 8-arm-PEG (weight average molecular weight 10 kDa) powder and dopamine hydrochloride were added into anhydrous DMF solvent according to a molar ratio of 1:4, and stirred at 40°C for 2h. After the reaction was completed, the reaction solution was poured into pre-iced anhydrous petroleum ether, and the product was filtered after precipitation. After washing with pure water, it was freeze-dried to obtain SG-PEG-DA. SG-PEG-DA (weight average molecular weight 10 kDa) and gold nanoparticles were dissolved in PBS (pH = 7.4) solution according to a molar ratio of 10:1, and stirred at 37°C for 24h. After the reaction was completed, the product was collected by centrifugation and freeze-dried to obtain Au-PEG-DA.

[0127] (2) Preparation of manganese nanoparticle-coated gold nanoparticles (Mn@AuNP)

[0128] Au-PEG-DA (weight average molecular weight 10 kDa) and BSA were dissolved in pure water solution according to a molar ratio of 2:1, followed by the addition of 2% KMnO4 by mass, and stirred at 40°C for 2h. After the reaction was completed, the product was collected by centrifugation, and finally the thiol-modified AS1411 nucleic acid aptamer was modified to obtain the nano-radiotherapy sensitizer. After freeze-drying, Mn@AuNP was obtained.

[0129] Example 4: Method 4 for preparing manganese nanoparticle-coated gold nanoparticles (Mn@AuNP)

[0130] (1) Preparation of gold nanoparticle sensitizer (Au-PEG-DA)

[0131] Under the protection of nitrogen, 8-arm-PEG (weight average molecular weight 40 kDa) powder and dopamine hydrochloride were added into anhydrous DMF solvent according to a molar ratio of 1:4, and stirred at 40°C for 2h. After the reaction was completed, the reaction solution was poured into pre-iced anhydrous petroleum ether, and the product was filtered after precipitation. After washing with pure water, it was freeze-dried to obtain SG-PEG-DA. SG-PEG-DA (weight average molecular weight 40 kDa) and gold nanoparticles were dissolved in PBS (pH = 7.4) solution according to a molar ratio of 10:1, and stirred at 37°C for 24h. After the reaction was completed, the product was collected by centrifugation and freeze-dried to obtain Au-PEG-DA.

[0132] (2) Preparation of manganese nanoparticle-coated gold nanoparticles (Mn@AuNP)

[0133] Au-PEG-DA (weight average molecular weight 40 kDa) and BSA were dissolved in pure water solution according to a molar ratio of 2:1, followed by the addition of 2% KMnO4 by mass, and stirred at 40°C for 2h. After the reaction was completed, the product was collected by centrifugation, and finally the thiol-modified AS1411 nucleic acid aptamer was modified to obtain the nano-radiotherapy sensitizer. After freeze-drying, Mn@AuNP was obtained.

[0134] Example 5: Method 5 for preparing gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0135] (1) Preparation of gold nanoparticle sensitizer (Au-PEG-DA)

[0136] Under nitrogen protection, 8-arm-PEG (weight average molecular weight 20 kDa) powder and dopamine hydrochloride were added to anhydrous DMF solvent according to a molar ratio of 1:4, and stirred at 40°C for 2h. After the reaction was completed, the reaction solution was poured into pre-iced anhydrous petroleum ether, and the product was filtered after precipitation. After washing with pure water, it was freeze-dried to obtain SG-PEG-DA. SG-PEG-DA (weight average molecular weight 20 kDa) and gold nanoparticles were dissolved in PBS (pH = 7.4) solution according to a molar ratio of 10:1, and stirred at 37°C for 24h. After the reaction was completed, the product was collected by centrifugation and freeze-dried to obtain Au-PEG-DA.

[0137] (2) Preparation of gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0138] Au-PEG-DA (weight average molecular weight 20 kDa) and BSA were dissolved in pure water solution according to a molar ratio of 4:1, and then 2% KMnO4 was added. The mixture was stirred at 40°C for 2h. After the reaction was completed, the product was collected by centrifugation. Finally, the thiol-modified AS1411 nucleic acid aptamer was used to modify the nanoradiotherapy sensitizer, and Mn@AuNP was obtained after freeze-drying.

[0139] Example 6: Method 6 for preparing gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0140] (1) Preparation of gold nanoparticle sensitizer (Au-PEG-DA)

[0141] Under nitrogen protection, 8-arm-PEG (weight average molecular weight 10 kDa) powder and dopamine hydrochloride were added to anhydrous DMF solvent according to a molar ratio of 1:4, and stirred at 40°C for 2h. After the reaction was completed, the reaction solution was poured into pre-iced anhydrous petroleum ether, and the product was filtered after precipitation. After washing with pure water, it was freeze-dried to obtain SG-PEG-DA. SG-PEG-DA (weight average molecular weight 10 kDa) and gold nanoparticles were dissolved in PBS (pH = 7.4) solution according to a molar ratio of 10:1, and stirred at 37°C for 24h. After the reaction was completed, the product was collected by centrifugation and freeze-dried to obtain Au-PEG-DA.

[0142] (2) Preparation of gold nanoparticle-coated manganese dioxide (Mn@AuNP)

[0143] Au-PEG-DA (weight average molecular weight 10 kDa) and BSA were dissolved in a pure water solution at a molar ratio of 4:1, followed by the addition of 2% KMnO4 by mass, and stirring at 40°C for 2h. After the reaction was completed, the product was collected by centrifugation, and finally the thiol-modified AS1411 nucleic acid aptamer was modified to the nano-radiotherapy sensitizer, and after freeze-drying, Mn@AuNP was obtained.

[0144] Comparative Example 1: Preparation of PEG-modified gold nanoparticles (Au-PEG)

[0145] 8-arm-SG-PEG (weight average molecular weight 10 kDa) and the prepared gold nanoparticles were dissolved in a PBS (pH = 7.4) solution at a molar ratio of 10:1, and stirred at 37°C for 24h. After the reaction was completed, the product was collected by centrifugation and freeze-dried to obtain PEG-modified gold nanoparticles (Au-PEG).

[0146] Example 7: Performance test of Mn@AuNP nanomaterial

[0147] In this embodiment, the TEM photo of the nanoparticles was measured by the Hitachi transmission electron microscope HT7800; the hydrated particle size was measured by the Malvern laser particle size instrument Mastersizer 3000; the Raman spectrum was measured by the Horiba laser confocal Raman instrument LabRAM HR Evolution; and the fluorescence microscope photo was measured by the Zeiss ApoTome.

[0148] 1. Transmission electron microscope test

[0149] The Mn@AuNP prepared in Example 6 and the Au-PEG prepared in Comparative Example 1 were subjected to transmission electron microscope analysis, and further observed for morphology and size. The results are shown in Figure 1 .

[0150] The results show that the nanomaterial prepared in Comparative Example 1 has a uniform morphology, which is circular, and the particle size is about 15nm; the nanomaterial prepared in Example 6 has a nanoflower shape, and the gold nanoparticles are dispersed on the surface of the manganese dioxide particles, and the Mn@AuNPs have a particle size of about 110nm.

[0151] 2. Hydrated particle size test

[0152] The Mn@AuNP prepared in Examples 1-6 were dispersed in pure water, respectively, and ultrasonicated for 10min to completely disperse the nanoparticles. The particle size of each nanoparticle was measured using a Malvern particle size instrument, and the particle size was measured three times. The test results are shown in Figure 2 .

[0153] Results show that increasing the molecular weight of 8-arm-PEG can significantly increase the particle size of the Mn@AuNP nanoparticles, and increasing the ratio of gold nanoparticles to BSA can also increase the particle size. The particle size of the nanoparticles prepared in Examples 1-6 can meet the requirements of targeting tumors, and the nanoparticles prepared in Example 6 are the most preferred.

[0154] 3. Surface-enhanced Raman performance test

[0155] The Mn@AuNP nanomaterial prepared in Example 6 and the Au-PEG material prepared in Comparative Example 1 were subjected to laser Raman confocal testing, and the test results are shown in Figure 3

[0156] Results show that Mn@AuNP significantly enhances the Raman characteristic peaks in tumor cells, and thus can be used for detection of tumor cells.

[0157] Example 8: Mn@AuNP increases intracellular ROS free radical levels

[0158] 4T1 cells were seeded in a 24-well plate at a density of 4x10 4 cells / well and cultured for 12 h until the cells were completely adherent. The cells were divided into two groups, one group was added with PBS, and the other group was added with Mn@AuNP prepared in Example 6, and cultured for 8 h, then irradiated with 4Gy X-ray. Subsequently, washed twice with PBS, added with 10 μM ROS fluorescent probe (DCFH-DA) and incubated for 30 min, washed twice with PBS again, and the green fluorescence distribution was observed by fluorescence microscopy. The results are shown in Figure 4

[0159] Results show that single radiotherapy can only promote a small amount of ROS free radicals, and under the action of the Mn@AuNP nanosensitizer, the generation of intracellular ROS free radicals is significantly increased.

[0160] Example 9: Mn@AuNP enhances in vitro killing effect on tumor cells

[0161] 4T1 cells were seeded in a 96-well plate at a density of 3x10 3 cells / well and cultured for 12 h until the cells were completely adherent. The cells were divided into three groups, each group was added with PBS, Au-PEG of Comparative Example 1, and Mn@AuNP of Example 6, respectively, and cultured for 8 h, then irradiated with 4Gy X-ray. Continue to culture for 24 h, add CCK-8 reagent, and use an enzyme marker to determine cell activity.

[0162] Results are shown in Figure 5 Results show that under X-ray irradiation, the nanosensitizer significantly kills tumor cells.

[0163] ​​Example 10: Mn@AuNP enhanced anti-tumor effect in vivo

[0164] 6-8 weeks old balb / c mice were selected, and 1 x 10 6 4T1 cells were injected into the mammary fat pad to establish 4T1 breast cancer model. When the tumor tissue grew to about 100 mm 3 around, the tumor-bearing mice were randomly divided into 4 groups, and each group was injected with PBS, Au-PEG of Comparative Example 1 and Mn@AuNP of Example 6 via tail vein, and was given 4Gy X-ray irradiation. The tumor tissue of mice was taken on the 14th day for photographing.

[0165] The results are shown in Table 1. Figure 6 The results show that the manganese dioxide radiotherapy sensitizing agent (D) wrapped by gold nanoparticles can significantly enhance the anti-tumor effect mediated by radiotherapy. Figure 6

[0166] All the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various changes and modifications to the preferred embodiments described herein will be apparent to those skilled in the art and can be made without departing from the scope of the application and that the scope of the application is not to be limited by the preferred embodiments set forth in the above examples.​

Claims

1. A gold nanocomposite, characterized by, The composite material comprises: (i) a manganese dioxide core; and (ii) gold nanoparticles distributed on the surface; wherein the gold nanoparticle composite material has one or more properties selected from the group consisting of: (1) an improved surface-enhanced Raman spectroscopy signal level; (2) an improved X-ray deposition efficiency; (3) an improved level of ROS free radicals in tumor cells; (4) an improved tumor killing effect in radiotherapy.

2. The gold nanocomposite of claim 1, wherein, The composite material further comprises: (iii) a targeting component on the surface of the gold nanoparticles.

3. A method of preparing the gold nanocomposite of claim 1, wherein, The method comprises the steps of: (a) reacting dopamine-grafted eight-arm polyethylene glycol with gold nanoparticles to obtain dopamine-polyethylene glycol modified gold nanoparticles; (b) subjecting the dopamine-polyethylene glycol modified gold nanoparticles obtained in step (a) to a redox reaction with potassium permanganate and bovine serum albumin to obtain the gold nanoparticle composite material of claim 1.

4. The method of claim 3, wherein, Step (a) comprises: (a1) adding eight-arm polyethylene glycol (8-arm-PEG) and dopamine hydrochloride to anhydrous polar solvent to react to form dopamine-grafted eight-arm polyethylene glycol (8-arm-PEG-DA); (a2) mixing and reacting the dopamine-grafted eight-arm polyethylene glycol (8-arm-PEG-DA) in step (a1) with gold nanoparticles to obtain dopamine-polyethylene glycol modified gold nanoparticles (Au-PEG-DA).

5. The method of claim 4, wherein, The molar ratio of dopamine-grafted eight-arm polyethylene glycol (8-arm-PEG-DA) to gold nanoparticles in step (a2) is (10-5):(2-1).

6. The method of claim 3, wherein, The molar ratio of dopamine-polyethylene glycol modified gold nanoparticles to bovine serum albumin in step (b) is (4-2):

1.

7. The method of claim 3, wherein, The method further comprises the step of: (c) modifying a targeting component to the surface of the product of step (b).

8. Use of the gold nanocomposite according to claim 1, characterized in that, A reagent for one or more uses selected from the group consisting of: (1) tumor radiotherapy sensitization; (2) tumor diagnosis; (3) tumor imaging; (4) identification and localization of tumor cells.

9. A tumor radiotherapy sensitizing agent, characterized by, The tumor radiotherapy sensitization agent comprises the gold nanoparticle composite material of claim 1.

10. A diagnostic reagent, characterized by, The diagnostic reagent comprises the gold nanoparticle composite material of claim 1.

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