125I nano implant particle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of brachytherapy and radiotherapy sensitization, and particularly provides a 125I nano implant particle and a preparation method and application thereof. The 125I nano implant particle is of a core-shell structure and comprises a particle inner core, 125I adsorbed on the outer surface of the particle inner core and a covalent organic framework shell which is coated outside the particle inner core and the 125I and contains an I-type photosensitizer. The inner core of the 125I nano implant particle is used for improving energy deposition of gamma rays and X rays and generating more hydroxyl radicals; the I-type photosensitizer-containing frame shell is used for absorbing the energy of Auger electrons and internal conversion electrons and exciting the photosensitizer to generate singlet oxygen, so that the utilization efficiency of 125I decay energy is effectively improved. The 125I nano implanted particles are combined with internal radiation radiotherapy and radiodynamic therapy to destroy tumor cell DNA and cell membranes, enhance tumor killing and induce immunogenic cell death, so that anti-tumor immunity is activated, and a remote isolation effect is induced.

Description

Technical Field

[0001] The present invention relates to the technical field of brachytherapy and radiotherapy sensitization, and specifically to a 125 I. Nano-implanted particles and their preparation method and application. Background Art

[0002] Radiotherapy is the first-line treatment for cancer, and more than 50% of cancer patients benefit from it. Depending on the type of radiation source, radiotherapy can be divided into two types: external radiation therapy, which uses external ionizing radiation (X-rays, gamma rays, heavy ion rays, etc.) to kill tumors; internal radiation therapy, which uses radionuclides ( 177 Lu, 225 Ac, 131 I. 125 I, etc.) decay in the body to release ionizing radiation that destroys tumors. Although external beam radiotherapy (mainly X-ray external beam radiotherapy) is becoming increasingly mature, the challenge of minimizing damage to normal tissues still exists. Precise and personalized internal beam radiotherapy is a new trend in the future development of radiotherapy. Brachytherapy is a type of internal beam radiotherapy. With the help of imaging techniques such as CT and ultrasound, the radiation source is precisely implanted into the tumor area according to the preoperative plan, and the decay of radioactive nuclides is used to continuously release ionizing radiation to kill the tumor. Unlike external beam radiotherapy, internal beam radiotherapy destroys the tumor from the inside, and the ionizing radiation does not pass through normal tissue, so the side effects on the body are even smaller. Traditional iodine seed sources generally refer to 125 I particles, 125 I particles are a commonly used interstitial brachytherapy technique in clinical practice in my country. 125 The substrate (such as a silver rod) and the titanium shell of I are sealed by welding. 125 I particles can continuously release 27.4keV to 31.4keV X-rays and gamma rays to kill tumors. Their effective radius in the tissue is 1.7cm, so they can deliver extremely high radiation doses to the tumor site while avoiding damage to normal tissues. Compared with external radiotherapy, radioactive particle implantation therapy overcomes the disadvantage that re-radiotherapy cannot increase the tumor dose. It has the characteristics of high local dose and less damage to normal tissues. It also has the advantages of less trauma and good patient tolerance. At present, 125 I particles are suitable for patients with various types of cancer, especially gynecological tumors and prostate cancer.

[0003] However, traditional close-range treatment technology is more convenient for single small tumors. As the tumor size increases and the number of lesions increases, the number of puncture needles and implanted particles required for surgery increases significantly, and the operation time is prolonged and the difficulty of the operation increases. On the one hand, it has led to a substantial increase in treatment costs. On the other hand, it has also made difficult operations only accessible to a few highly skilled physicians. As the operation time increases, the patient's tolerance decreases significantly. Weak patients cannot tolerate the treatment and lose the opportunity for surgery. Therefore, it is necessary to further optimize the treatment efficiency of traditional iodine seed sources, and exploring new, efficient, and easier-to-operate iodine seed sources has become an urgent clinical need.

[0004] To this end, the present invention is proposed. Summary of the invention

[0005] The main purpose of the present invention is to provide a 125 I nanometer implanted particles and their preparation method and application, the present invention 125 Nanoparticles are designed as core-shell structures to more efficiently utilize 125 I decay releases radiation energy. Covalent organic frameworks containing type I photosensitizers are used as 125 The outer shell of the nanoparticle is used to absorb the energy of Auger electrons and internal conversion electrons and excite the photosensitizer molecules to produce singlet oxygen ( 1 O2), effectively improve 125 I The utilization efficiency of decay energy.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions.

[0007] A first aspect of the present invention provides a 125 1 nanometer implant particle, 125 The nanometer implanted particle has a core-shell structure, which includes an implanted particle core, and a 125 I, and coated on the implanted particle core and the 125 I external covalent organic framework shell; the covalent organic framework shell is a covalent organic framework shell containing type I photosensitizer.

[0008] In some embodiments of the present invention, the type I photosensitizer-containing covalent organic framework shell comprises a porphyrin-containing organic framework shell.

[0009] Preferably, the thickness of the covalent organic framework shell is 10 nm to 50 nm.

[0010] In some embodiments of the present invention, the core of the implanted particle is a core-shell structure, which includes a nano core particle and a silver shell coated on the outside of the nano core particle; 125 I is adsorbed on the outer surface of the silver shell.

[0011] Preferably, the radius of the nano core particle is 25 nm to 50 nm, and the thickness of the silver shell is 3 nm to 10 nm.

[0012] In some embodiments of the present invention, the nano core particles include at least one of gold nanoparticles and heavy metal particles.

[0013] Preferably, the heavy metal particles are selected from at least one of bismuth sulfide, hafnium oxide and nano-platinum.

[0014] The second aspect of the present invention provides a 125 A method for preparing nanometer implanted particles, the method comprising the following steps:

[0015] preparing implantable particle cores;

[0016] The implanted particle core after the first surface modification is subjected to a covalent organic framework synthesis reaction to obtain an implanted particle core@covalent organic framework shell;

[0017] After the implanted particle core@covalent organic framework shell is subjected to a second surface modification, the implanted particle core@covalent organic framework shell is placed in a 125 I solution was allowed to stand, and the obtained solution was centrifuged and washed. 125 I nanometer implant particles.

[0018] In some embodiments of the present invention, the 125 In the solution of I 125 I activity is 50μCi~500μCi. 100μg is implanted into the particle core @ covalent organic framework shell and 50μCi~500μCi is added 125 I, for example, can be one of 50μCi, 80μCi, 100μCi, 150μCi, 200μCi, 250μCi, 300μCi, 350μCi, 400μCi, 450μCi, 500μCi or any value that satisfies the above range.

[0019] In some embodiments of the present invention, 125 Solutions of I include sodium iodide solutions.

[0020] In some embodiments of the present invention, the implanted particle core comprises an Au@Ag core,

[0021] Under acidic conditions and inert gas protection, the Au@Ag core after the first surface modification is reacted with 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin and 2,5-dihydroxyterephthalaldehyde at 120° C. in the dark for 24 h to 72 h to obtain Au@Ag@TAPP-COF.

[0022] In some embodiments of the present invention, the second surface modification uses a solution containing polyethylene glycol; and the first surface modification uses a solution containing polyvinyl pyrrolidone.

[0023] In some embodiments of the present invention, the preparation of the Au@Ag core comprises:

[0024] The Au@Ag core particles are mixed with a solution containing polyethyleneimine and a solution containing ascorbic acid, and a solution containing silver ions is added dropwise until the color of the solution changes to orange-red, thereby obtaining the Au@Ag core.

[0025] The third aspect of the present invention provides a 125 I nanometer implanted particles or the preparation method of the second aspect 125 Application of nanoparticle implants in brachytherapy.

[0026] Compared with the prior art, the present invention achieves the following technical effects:

[0027] 1. In the present invention 125 Nanoparticles are designed as core-shell structures to more efficiently utilize 125 I decay releases radiation energy. Organic framework containing type I photosensitizer as 125 The outer shell of the nanoparticle is used to absorb the energy of Auger electrons and internal conversion electrons and excite the photosensitizer molecules to produce singlet oxygen ( 1 O2).

[0028] 2. In the present invention 125 Nanoparticle implants as radiosensitizers in improving 125 Based on the radiotherapy effect of I particles themselves, the traditional 125 I particles cannot be used 125 The electron energy generated by I decay (AEs and ICEs) is converted into cytotoxic 1 O2, effectively improving 125 I The utilization efficiency of decay energy.

[0029] 3. In the present invention, gold nanoparticles are used as 125 I nanoparticles are implanted into the core of the particle, facilitating the energy deposition of X-rays and gamma-rays to enhance the radiolysis of water to produce additional hydroxyl radicals (·OH).

[0030] 4. In the present invention 125 I nanoparticles combined with internal irradiation and radiodynamic therapy can destroy the DNA and cell membrane of tumor cells to achieve the effect of radiation and reactive oxygen species (·OH and 1O2) has a dual tumor killing effect, which greatly improves the killing of tumor cells; at the same time, the destruction of cell membrane integrity will lead to the release of intracellular mediators, thereby activating anti-tumor immunity and showing a certain systemic therapeutic effect in animal models, thereby achieving local treatment-induced remote effects and expanding the application of close-range radiotherapy in cancer treatment.

[0031] 5. In the present invention 125 The particle size of the nanoparticles is about 130 nanometers, which can be well dispersed in saline, allowing them to be delivered to the tumor area through a long needle, performing less invasive surgery. Good dispersibility also facilitates uniform dose deposition in the tumor area, reducing the radiation dose required for treatment and the difficulty of operation.

[0032] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0034] Figure 1 for 125 I nanoparticle implants ( 125 Preparation process flow chart of I-Au@Ag@TAPP);

[0035] Figure 2 for 125 I nanoparticle implants ( 125 Transmission electron microscopy image of I-Au@Ag@TAPP);

[0036] Figure 3 It is the hydroxyl radical yield diagram under different conditions;

[0037] Figure 4 is a graph of singlet oxygen yield under different conditions;

[0038] Figure 5 for 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) uptake diagram of 4T1 cells;

[0039] Figure 6 for 125 I nanoparticle implants ( 125I-Au@Ag@TAPP) cell killing effect on 4T1 and B16F10 cells;

[0040] Figure 7 for 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) induced cell membrane lipid peroxidation.

[0041] Figure 8 for 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) induced cell CRT exposure diagram;

[0042] Fig. 9 for 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) induced cell release of HMGB1;

[0043] Fig.10 for 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) was used to treat bilateral tumors in mice, and combined with immunotherapy to induce remote effects. DETAILED DESCRIPTION

[0044] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of the concept of the present invention.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the specification and claims of the present invention and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0046] In the description of the embodiments of the present invention, the technical terms "first", "second", etc. are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features. In the description of the embodiments of the present invention, the meaning of "multiple" is more than two, unless otherwise clearly and specifically defined.

[0047] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0048] In the description of the embodiments of the present invention, the term "and / or" is only a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0049] In the description of the embodiments of the present invention, the term "multiple" refers to more than two (including two). Similarly, "multiple groups" refers to more than two groups (including two groups), and "multiple pieces" refers to more than two pieces (including two pieces).

[0050] Traditional 125 I particles have good therapeutic effects on single small tumors and are suitable for various types of cancer patients, especially gynecological tumors and prostate cancer. 125 I particles are facing many challenges:

[0051] (1) 125 I nuclides produce gamma rays, X-rays, Auger electrons (AEs, 12.2keV) and internal conversion electrons (ICEs, 7.2keV) through electron capture decay, of which the energy of electrons (AEs and ICEs) accounts for ~32% of the total decay energy (61.4keV). However, these electrons have a short range (AEs range: 1.5nm~14.0μm, ICEs range: 0.7~16μm) and are difficult to penetrate. 125 I-particle titanium shell (50 μm), therefore, conventional 125 I-particles are not fully utilized 125 I decay produces radiation energy.

[0052] (2) Traditional 125 I-particle implantation is a local treatment mode that has a therapeutic effect only on the tumor area where the particles are implanted. Clinically, no significant regression of tumor lesions in areas where particles are not implanted has been observed, and more than 90% of cancer-related deaths are caused by cancer metastasis. As the tumor volume increases and the number of lesions increases, 125The number of puncture needles and implanted particles required for I-seed implantation surgery increases significantly, the operation time is prolonged, the operation difficulty increases, and the treatment effect is limited. On the one hand, it leads to a substantial increase in treatment costs. On the other hand, high-difficulty surgery can only be performed by a few highly skilled physicians. As the operation time increases, the patient's tolerance decreases significantly. Weak patients cannot tolerate the treatment and lose the opportunity for surgery.

[0053] (3) Traditional 125 I particles are implanted into the tumor area through catheter puncture. This procedure is somewhat invasive and is gradually being replaced by other more advanced treatment modes.

[0054] Nanoparticles containing high atomic number atoms have been used as radiotherapy sensitizers. Compared with biological tissues, high atomic number atoms are more likely to interact with ionizing radiation, thereby enhancing the deposition of radiation energy in the tumor area and improving the tumor-killing effect of radiotherapy. Among them, hafnium dioxide (HfO2) nanoparticles have been clinically approved and have shown good enhanced radiotherapy effects after local injection in the tumor area. In recent years, nano-brachytherapy using nano-radiotherapy sensitizers as radionuclide carriers has attracted the attention of researchers. Compared with traditional brachytherapy, nano-brachytherapy improves the treatment effect while facilitating less invasive surgery. Therefore, exploring 125 Highly efficient radiosensitizers for I are expected to optimize its clinical application value. 125 I decay produces X-rays, gamma rays, Auger electrons and internal conversion electrons. The energy of Auger electrons and internal conversion electrons accounts for about 32% of the total decay energy, but these electrons with high energy transfer line density are 125 The titanium wall of the I particle absorbs the radiation energy, resulting in a waste of radiation energy. 125 The Auger electrons generated by I decay have an average energy of about 500 eV and can deposit a large amount of radiation energy within their nanoscale trajectories (<50 nanometers), which is well matched to the size of nanomaterials. 125 The electron energy that cannot be used by I particles is to increase 125 I particles are one of the key factors in the effectiveness of tumor treatment.

[0055] After a tumor lesion is irradiated with radiotherapy, other lesions far from the radiotherapy site also shrink, which is called the bystander effect or abscopal effect. It is generally believed that this phenomenon is related to the release of tumor-related antigens in the irradiated site after radiotherapy, which reactivates the immune system and breaks immune tolerance. Because its incidence is very low and difficult to predict, there were only individual case reports in the early days and it did not attract much attention. It was not until the past 20 years when radiotherapy technology has been continuously improved, especially after the widespread application of stereotactic body radiation therapy (SBRT), that reports of abscopal effects have continued to increase, which has once again attracted the attention of the oncology community. Tumors with strong immunogenicity, such as melanoma and renal cancer, have a higher incidence of abscopal effects. In particular, the exploration in melanoma has been the most successful. In a retrospective study, Grimaldi et al. reported that the incidence of abscopal effects in 21 patients treated with ipilimumab combined with radiotherapy was 52%. Compared with patients without abscopal effects, the median survival of patients with abscopal effects increased from 8.3 months to 22.4 months. Currently, the consensus in the oncology community is that combining radiotherapy with immunotherapy can increase the probability of abscopal effects.

[0056] Photodynamic therapy (PDT) is a clinically approved, almost non-invasive cancer treatment option. Typically, external light is used to activate photosensitizers (such as porphyrins) to generate cytotoxic reactive oxygen species (the most important of which is singlet oxygen, 1 O2) kills cancer cells. In addition, 1 O2 is immunogenic and can induce immunogenic cell death in tumor cells, thereby activating anti-tumor immunity. In a large number of animal model studies, photodynamic therapy can induce remote effects. However, due to the rapid attenuation of light in biological tissues and the limitation of tissue penetration, the clinical application of photodynamic therapy is usually limited to surface tissues, such as the treatment of skin cancer. How to achieve photodynamic therapy for deep tumors is a long-standing problem in clinical practice.

[0057] In recent years, radiodynamic therapy (RDT) has been favored by researchers. This treatment mode uses nuclear radiation to excite photosensitizers to produce cytotoxic reactive oxygen species, combining radiotherapy and photodynamic therapy to improve the anti-tumor effect of radiotherapy. Nuclear radiation has good tissue penetration and is currently widely used in the diagnosis and treatment of cancer. Using radiation as an excitation source for photodynamic therapy can not only fully retain the high clinical relevance of photodynamic therapy, but also break through the depth limitation of traditional photodynamic therapy and achieve photodynamic therapy for deep tumors. In particular, RDT that produces singlet oxygen can induce immunogenic cell death and activate anti-tumor immunity, achieving significant inhibition of tumor growth at the non-irradiated end (remote effect). The generation of singlet oxygen may originate from the excitation of photosensitizer molecules (such as porphyrins) in radiotherapy sensitizers by Auger electrons.

[0058] In summary, the present invention has developed a method for brachytherapy. 125 I nanometer implant particles. 125 I decay produces X-rays, gamma rays, Auger electrons and internal conversion electrons. On the one hand, 125 The nano-core of the nano-implanted particles can enhance the long-range photon energy deposition (X-rays, γ-rays), promoting the radiolysis of water to produce more ·OH; on the other hand, 125 The photosensitizer shell of the nanoparticles can be used to 125 The energy of Auger electrons and internal conversion electrons that cannot be used by I particles excites the photosensitizer, producing a more wide-ranging and immunogenic 1 O2. 125 Nanoparticle implants combined with brachytherapy and radiodynamic therapy enhance 125 The tumor treatment effect of I particles and the anti-tumor immune activation of the whole province are expected to expand 125 Prospects of clinical application of I particles.

[0059] One aspect of the present invention provides a Figure 2 shown 125 I nanometer implant particles, the 125 The nanometer implanted particles have a core-shell structure, which includes an implanted particle core, and a 125 I, and coated on the implanted particle core and 125 The covalent organic framework shell is a covalent organic framework shell containing a type I photosensitizer.

[0060] In some embodiments of the present invention, 125 Nanoparticles are designed as core-shell structures to more efficiently utilize 125 The radiation energy released by the decay of type I. The covalent organic framework shell containing type I photosensitizer is used as 125The outer shell of the nanoparticle is used to absorb the energy of Auger electrons and internal conversion electrons and excite the photosensitizer molecules to produce singlet oxygen ( 1 O2).

[0061] 125 Nanoparticle implants as radiosensitizers in improving 125 Based on the radiotherapy effect of I particles themselves, the traditional 125 I particles cannot be used 125 The electron energy generated by I decay (AEs and ICEs) is converted into cytotoxic 1 O2, effectively improving 125 I The utilization efficiency of decay energy.

[0062] In some embodiments of the present invention, the covalent organic framework shell containing a type I photosensitizer may be a porphyrin-containing organic framework shell.

[0063] In some embodiments of the present invention, the covalent organic framework containing porphyrin is used as 125 The outer shell of the nanoparticle is used to absorb the energy of Auger electrons and internal conversion electrons and excite porphyrin molecules to produce singlet oxygen ( 1 O2).

[0064] In some embodiments of the present invention, the photosensitizer in the covalent organic framework shell containing type I photosensitizer includes but is not limited to 4,4'-(porphyrin-5,15-diyl)diphenylamine, tetraaldehyde phenylporphyrin, 2,6-diiodo BODIPY, dihydrochlorin, hematoporphyrin derivatives, hematoporphyrin monomethyl ether, hypocrellin A, hypericin, curcumin, phthalocyanine, and 5-aminolevulinic acid.

[0065] It should be noted that in the embodiments of the present invention, the covalent organic framework shell containing type I photosensitizer can also be obtained by post-modification and adsorption of photosensitizer molecules, for example, photosensitizer molecules are introduced into the obtained organic framework shell by post-modification, adsorption, etc.

[0066] In some embodiments of the present invention, the thickness of the covalent organic framework shell is 10 nm to 50 nm.

[0067] Exemplarily, the thickness of the covalent organic framework shell can be one of 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, or any value satisfying the above range.

[0068] In some embodiments of the present invention, the core of the implanted particle is a core-shell structure, which includes a nano core particle and a silver shell coated on the outside of the nano core particle; 125 I is adsorbed on the outer surface of the silver shell.

[0069] In some embodiments of the present invention, the radius of the nano core particle is 25 nm to 50 nm, and the thickness of the silver shell is 3 nm to 10 nm.

[0070] Illustratively, the radius of the nano core particle may be one of 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, or any value satisfying the above range.

[0071] Exemplarily, the thickness of the silver shell may be one of 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, or any value satisfying the above range.

[0072] In some embodiments of the present invention, 125 In the nano-implanted particles, the radius of the nano-core particle, the thickness of the silver shell and the thickness of the covalent organic framework outer shell are all adjustable, among which the thickness of the covalent organic framework outer shell will significantly affect the yield of singlet oxygen.

[0073] In some embodiments of the present invention, the nanocore particles include but are not limited to gold nanoparticles and heavy metal particles.

[0074] In some embodiments of the present invention, the nanocore particles are 125 I nanoparticles are implanted into the core of the particle, facilitating the energy deposition of X-rays and gamma-rays to enhance the radiolysis of water to produce additional hydroxyl radicals (·OH).

[0075] For example, gold nanoparticles are used as 125 I nanoparticles are implanted into the core of the particle, facilitating the energy deposition of X-rays and gamma-rays to enhance the radiolysis of water to produce additional hydroxyl radicals (·OH).

[0076] In some embodiments of the present invention, the heavy metal particles include, but are not limited to, bismuth sulfide, hafnium oxide, and nano-platinum.

[0077] Another aspect of the present invention provides a 125 I. Method for preparing nanometer implant particles.

[0078] Reference Figure 1 As shown, 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) is taken as an example to illustrate its preparation method.

[0079] Preparation of gold nanoparticles: 5mL-10mL 1wt% tetrachloroauric acid aqueous solution, 5mL-15mL gold seed sol and 1mL-2mL 2wt% polyethyleneimine (MW: 10000) aqueous solution were mixed, and the color of the solution changed from light red to orange-yellow. After stirring at room temperature for 3 minutes, 5mL-10mL (equal to the volume of tetrachloroauric acid aqueous solution) and 0.03mol / L hydroquinone aqueous solution were quickly added under rapid stirring, and the reaction was continued for 30 minutes to obtain a deep red gold nanoparticle sol, which was refrigerated at 4°C away from light for later use.

[0080] Preparation of Au@Ag nanoparticles: 500mL of gold nanoparticle sol, 10mL-20mL of 2wt% polyethyleneimine (MW: 10000) aqueous solution and 5mL of 0.1mol / L ascorbic acid aqueous solution were mixed, stirred at room temperature, and 5mL-10mL of 0.01mol / L silver nitrate solution was slowly added dropwise within 10min, and the color of the solution gradually changed from dark red to orange-red. The reaction was continued with stirring for 30min to obtain orange-red Au@Ag nanoparticle sol, which was refrigerated at 4°C away from light for later use.

[0081] Preparation of Au@Ag@TAPP: 500mL of Au@Ag nanoparticle sol was collected by centrifugation, washed twice with ethanol and n-butanol, and then resuspended in 5mL~10mL n-butanol. 5mL~10mL o-dichlorobenzene (n-butanol: o-dichlorobenzene volume ratio = 1:1) and 100mg~400mg polyvinylpyrrolidone (concentration: 10mg / mL~20mg / mL) (MW: 58000) were added, and ultrasonic treatment was performed for 1h. 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin (TAPP, 3.7mg~22.5mg) and 2,5-dihydroxyterephthalaldehyde (DHTA, 2.0mg~12.3mg) (TAPP:DHTA mass ratio = 75 / 41) were added to the reaction solution, ultrasonic dispersion was performed for 10min, and 100μL acetic acid was added, and the mixture was kept in the dark for 4h. Then, 300 μL of acetic acid was added, and the mixture was degassed by freeze-pump-thaw cycles three times. The tube was sealed with argon and heated at 120°C in the dark for 24 to 72 hours. After cooling to room temperature, the precipitate was separated by centrifugation, and the precipitate was washed with tetrahydrofuran and acetone in sequence until the supernatant was colorless. The product was resuspended in ethanol and stored in a dark place at 4°C for later use.

[0082] Polyethylene glycol modification of Au@Ag@TAPP: 5 mg Au@Ag@TAPP and 25 mg DSPE-PEG2000 were dispersed in 1 mL dimethyl sulfoxide (Au@Ag@TAPP: DSPE-PEG2000 mass ratio = 1:5, Au@Ag@TAPP concentration was 5 mg / mL), and ultrasonic treatment was performed for at least 1 h. The mixture was slowly added dropwise to 10 mL of vigorously stirred water (the volume of water was 10 times that of dimethyl sulfoxide) within 10 min. After stirring for about 30 min, the residual DSPE-PEG2000 and dimethyl sulfoxide were removed by dialysis with deionized water (molecular weight cutoff was 10 kDa), and the product was resuspended in deionized water by centrifugation to obtain polyethylene glycol-modified Au@Ag@TAPP, which was refrigerated at 4 °C away from light for later use.

[0083] It is worth mentioning that DSPE-PEG2000 can be replaced by FITC-DSPE-PEG2000 (partial replacement, such as 20%) to prepare fluorescein-PEG-modified Au@Ag@TAPP according to a similar procedure.

[0084] preparation 125 I-Au@Ag@TAPP: Take Au@Ag@TAPP (100 μL, 1 mg / mL) in a 1.5 mL centrifuge tube and add Na 125 I aqueous solution (50 μCi-500 μCi), 6 parallel groups, stand at room temperature for 5 min, 10 min, 20 min, 30 min, 60 min, 90 min, respectively, centrifuge and separate the precipitate, wash 3 times with deionized water, and resuspend the labeled product in 100 μL deionized water.

[0085] Labeling rate = product activity / 100 μCi×100%.

[0086] Another aspect of the present invention provides a 125 I nanometer implanted particles or prepared by the preparation method 125 Application of nanoparticle implants in brachytherapy.

[0087] In an embodiment of the present invention, 125 Nano-implanted particles are used in close-range radiotherapy to improve the efficacy of tumor treatment.

[0088] The present invention will be described in detail below with reference to specific embodiments. 125 I nanometer implant particles and their preparation method are described in detail.

[0089] If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0090] Example 1

[0091] Example 1 proposes a method for brachytherapy 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) can improve the therapeutic effect of tumors.

[0092] Preparation of gold seeds

[0093] Add 100mL of deionized water and 1mL of 1wt% tetrachloroauric acid aqueous solution to a 250mL two-necked flask, stir at 1000rpm, heat at 130℃, and reflux until violent boiling. Add 3mL of freshly prepared 1% (W / V) sodium citrate aqueous solution (reducing agent). The color of the solution changes from light yellow to gray, purple, and dark red in sequence. After the color stabilizes, continue stirring the reaction for 30min to obtain a gold seed sol, which is refrigerated at 4℃ away from light for later use.

[0094] Preparation of gold nanoparticles

[0095] In a 1L round-bottom flask, 475mL of deionized water, 10mL of 1wt% tetrachloroauric acid aqueous solution, 15mL of gold seed sol, and 1mL of 2wt% polyethyleneimine PEI (weight average molecular weight MW: 10000) aqueous solution were added in sequence, and the color of the solution changed from light red to orange-yellow. After stirring at room temperature for 3 minutes, 10mL of freshly prepared 0.03mol / L hydroquinone (HQ) aqueous solution was quickly added under rapid stirring, and the reaction was continued for 30 minutes to obtain a deep red Au nanoparticle sol, which was refrigerated at 4°C away from light for later use.

[0096] Preparation of Au@Ag nanoparticles

[0097] In a 1L round-bottom flask, 500mL of Au nanoparticle sol, 20mL of 2wt% polyethyleneimine (MW: 10000) aqueous solution, and 5mL of 0.1mol / L ascorbic acid aqueous solution were added in sequence, stirred and mixed at room temperature, and 10mL of 0.01mol / L silver nitrate solution was slowly added dropwise within 10min, and the color of the solution gradually changed from dark red to orange-red. The reaction was continued with stirring for 30min to obtain orange-red Au@Ag nanoparticle sol, which was refrigerated at 4℃ away from light for later use.

[0098] Preparation of Au@Ag@TAPP

[0099] Take 500mL of Au@Ag nanoparticle sol, collect it by centrifugation, wash it twice with ethanol and n-butanol, resuspend it in 5mL n-butanol, add 5mL o-dichlorobenzene and 200mg polyvinylpyrrolidone (PVP, MW: 58000), and ultrasonically treat it for 1h. The reaction solution was transferred to a 25mL Schlenk bottle, 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin (TAPP, 7.5mg, 0.011mmol) and 2,5-dihydroxyterephthalaldehyde (DHTA, 4.1mg, 0.025mmol) were added, and after ultrasonic dispersion for 10min, 100μL of acetic acid (AcOH) was added and it was kept in dark for 4h. Then 300μL of acetic acid was added, and after freeze-pump-thaw cycle degassing three times, the tube was sealed with argon gas and heated at 120℃ in dark for 72h. After cooling to room temperature, the precipitate was separated by centrifugation, and the precipitate was washed with tetrahydrofuran and acetone in sequence until the supernatant was colorless. The product was resuspended in 10 mL of ethanol and refrigerated at 4°C away from light for later use.

[0100] Polyethylene glycol modification of Au@Ag@TAPP

[0101] 5 mg Au@Ag@TAPP and 25 mg distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were dispersed in 1 mL dimethyl sulfoxide and ultrasonically treated for 1 h. The mixture was slowly added dropwise to 10 mL of vigorously stirred water within 10 min. After stirring for 30 min, the residual DSPE-PEG2000 and dimethyl sulfoxide were removed by dialysis with deionized water (molecular weight cutoff of 10 kDa). The product was resuspended in 5 mL of deionized water by centrifugation to obtain polyethylene glycol-modified Au@Ag@TAPP, which was refrigerated at 4°C away from light for later use.

[0102] preparation 125 I-Au@Ag@TAPP

[0103] Take Au@Ag@TAPP (100 μL, 1 mg / mL) in a 1.5 mL centrifuge tube and add Na 125 I aqueous solution (10 μL, 100 μCi), stand at room temperature for 60 min, centrifuge to separate the precipitate, wash three times with deionized water, and resuspend the labeled product in 100 μL deionized water.

[0104] Labeling rate = product activity / 100 μCi×100%.

[0105] Example 2

[0106] Example 2 adopts the same preparation process as Example 1, the only difference being that in the polyethylene glycol modification step, 20 wt % DSPE-PEG2000 is replaced with fluorescein polyethylene glycol phospholipid (FITC-DSPE-PEG2000) to obtain fluorescein-polyethylene glycol-modified Au@Ag@TAPP.

[0107] The following will be tested by experiment to obtain the 125 The performance of I-Au@Ag@TAPP was verified.

[0108] one, 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) produces ·OH and 1 O2

[0109] 1. Detection of OH: Prepare a 20 μmol / L APF (hydroxyl radical fluorescent probe, Thermo Fisher) 125 I-Au@Ag@TAPP, TAPP+Na 125 I. Na 125 I solution (solution concentration is 50ug / mL, 125 I activity concentration is 200 μCi / mL), and after standing in the dark for 12 h, 24 h, 36 h, and 48 h, the sample solution was centrifuged (15000×g, 3 min), the supernatant was collected, and the fluorescence signal intensity (488 nm excitation, 515 nm emission) was measured respectively.

[0110] The results are as follows Figure 3 As shown, 125 I-Au@Ag@TAPP can produce more 125 I. Na 125 I solution more ·OH.

[0111] 2. 1 O2 detection: 20 μmol / L SOSG (singlet oxygen fluorescence probe, Thermo Fisher) was prepared 125 I-Au@Ag@TAPP, TAPP+Na 125 I. Na 125 I solution (solution concentration is 50ug / mL, 125 I activity concentration is 200 μCi / mL), and after standing in the dark for 1 h, 2 h, 3 h, 4 h, and 5 h, the sample solution was centrifuged (15000×g, 3 min), the supernatant was collected, and the fluorescence signal intensity (504 nm excitation, 524 nm emission) was measured respectively.

[0112] The results are as follows Figure 4 As shown,125 I-Au@Ag@TAPP can significantly produce 1 O2, and TAPP+Na 125 I. Na 125 Solution I was almost undetectable 1 O2.

[0113] two, 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) 1 The main energy of O2 comes from Auger electrons

[0114] 125 The density of I-Au@Ag@TAPP was set to 1.0 g / cm 3 It is set as a three-layer structure, which consists of a spherical gold core with a radius of 40nm, a 3nm thick silver shell on the surface, and a TAPP covalent organic framework (COF) with a radius of 25nm.

[0115] Monte Carlo simulation was performed using Geant 4.10.7 software. 125 The electrons or photons produced by I decay 125 I-Au@Ag@TAPP interaction. The particle source that generates electrons or photons isotropically is placed evenly at 0.1 nm from the surface of the silver shell, and the external space is filled with water. The energy deposition at different locations is calculated. The results are shown in Table 1.

[0116] Table 1

[0117]

[0118] The results show that the ionizing radiation energy deposited in the TAPP-COF shell is mainly contributed by Auger electrons, proving that the excitation of TAPP produces 1 The main energy source of O2 comes from 125 Auger electrons released by I decay.

[0119] three, 125 I nanoparticle implants ( 125 Cellular uptake and cytotoxicity of I-Au@Ag@TAPP)

[0120] 1. Cell uptake: 4T1 cells were seeded in a 24-well plate (5×104 per well). After culturing for 12 h, the culture medium was discarded and culture medium containing 100 μg / mL Au@Ag@TAPP was added. After incubation for 1 h, 2 h, 4 h, and 8 h, the cells were washed three times with phosphate buffered saline (PBS). Trypsin solution was added to separate the cells from the culture dish, the cell suspension was collected, the supernatant was discarded by centrifugation, and the cells were digested with nitric acid (60°C, 24 h). The cells were diluted to 5 mL with deionized water and the Au content was determined by inductively coupled plasma-mass spectrometry.

[0121] The results are as follows Figure 5 As shown, the uptake of Au@Ag@TAPP by 4T1 cells increased over time.

[0122] 2. Cytotoxicity: 4T1 and B16F10 cells were seeded in 96-well plates (1×10 4 ), after culturing for 12 h, the culture medium was discarded and 50 μg / mL of 125 The culture medium was incubated with I-Au@Ag@TAPP (0 μCi / mL, 50 μCi / mL, 100 μCi / mL, 150 μCi / mL, and 200 μCi / mL). After incubation for 48 h, the cell viability was detected using the standard CCK8 method, and the absorbance at 450 nm was measured. The untreated cell population was used as a reference to establish 100% cell viability.

[0123] The results are as follows Figure 6 As shown, with 125 The increase in activity concentration of I, 125 The killing ability of I-Au@Ag@TAPP on 4T1 cells gradually increased.

[0124] Four, 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) induces immunogenic cell death

[0125] 1. Cell membrane lipid peroxidation: 4T1 cells were inoculated in a 35 mm confocal dish (2 × 105 / mL), the culture medium was discarded after 12 h of culture, and a 2% spherical nanoparticle containing Au@Ag@TAPP, Na 125 I. 125 I-Au@Ag@TAPP (material concentration is 50ug / mL, 125The cells were cultured in a medium containing 100 μCi / mL of 1 μmol / L of 1 μmol / L lipid peroxidation sensor (BODIPY 581 / 591C11) and then washed 3 times with PBS. The cells were then cultured for 1 hour and washed 3 times with PBS. The fluorescence intensity at 591 nm and 510 nm was quantitatively analyzed using Image J software.

[0126] The results are as follows Figure 7 As shown, 125 I-Au@Ag@TAPP-treated cells exhibited significant cell membrane lipid peroxidation compared with other control groups.

[0127] 2. Calreticulin (CRT) exposure: 4T1 cells were seeded in a 35 mm confocal dish (2×105 / mL), and the culture medium was discarded after 12 h of culture. 125 I. 125 I-Au@Ag@TAPP (material concentration of 50ug / mL, 125I activity concentration of 100μCi / mL) culture medium was added, and the culture medium was discarded after 24h of culture, and the cells were washed 3 times with PBS. The cells were blocked with PBS containing 1% BSA at 4℃ for 1h, and the supernatant was discarded; Alexa Fluor 488-CRT antibody (500 times diluted in PBS containing 1% BSA) was added and incubated at 4°C for 12 h. After washing with PBS three times, the cell nuclei were stained with DAPI, and the slides were sealed with anti-fluorescence quencher and photographed under a confocal fluorescence microscope. The fluorescence intensity was quantitatively analyzed using Image J software.

[0128] The results are as follows Figure 8 As shown, 125 I-Au@Ag@TAPP-treated cells showed significant CRT exposure compared with other control groups.

[0129] 3. High mobility group protein B1 (HMGB1) release: 4T1 cells were seeded in 96-well plates (1×104 per well), and the culture medium was discarded after 12 h of culture. 125 I. 125 I-Au@Ag@TAPP (material concentration is 50ug / mL, 125 The cells were cultured in a medium containing 100 μCi / mL of HMGB1. After culturing for 24 h, the HMGB1 concentration in the cell supernatant was determined according to the instructions of the ELISA kit.

[0130] The results are as follows Fig. 9 As shown, 125I-Au@Ag@TAPP-treated cells showed significant release compared with other control groups.

[0131] Auger electrons can be used to excite photosensitizers to produce singlet oxygen. 125 Nano-implanted particles will transform traditional 125 The unusable electron energy of the implanted particles (mainly Auger electrons) is converted into cytotoxic and immunogenic singlet oxygen, which enhances cell killing and induces immunogenic cell death.

[0132] five, 125 I nanoparticle implants ( 125 I-Au@Ag@TAPP) induces abscopal effect in a mouse bilateral tumor model

[0133] 1. Establishment of mouse tumor model: Female BALB / c mice (6-8 weeks old) were subcutaneously injected with 1×106 4T1 cells in PBS suspension (primary tumor) in the right shoulder, and 5 days later, 5×105 4T1 cells in PBS suspension (distal tumor) were subcutaneously injected in the left shoulder. The length (L) and width (W) of the tumor were measured with a digital vernier caliper. The tumor volume (V) was calculated according to the formula V = 1 / 2×L×W2.

[0134] 2. Treatment experiment: When the volume of the primary tumor reaches ~50mm 3 The mice were randomly divided into 3 groups (n=10) and injected with PBS (40 μL), 125 I-Au@Ag@TAPP-PEG, Au@Ag@TAPP (50 mg / kg, 1 mCi) and orthotopic implantation 125 I particles (0.5mCi×2), 5 mice were randomly selected from each group after treatment, and anti-PD-L1 antibody (75μg / mouse) was intraperitoneally injected every 3 days. Tumor volume and mouse weight were recorded every two days.

[0135] The results are as follows Fig.10 As shown, 125 I Nanoparticles Implanted for Tumor Treatment is Far Better Than Traditional 125 I implanted particles, combined with immunotherapy to activate systemic anti-tumor immunity, has obvious distal effects, while traditional 125 Implanted particles have almost no distal effects.

[0136] Combination immunotherapy can improve 125 The anti-tumor immune effect of nano-implanted particles is combined with PD-L1 immune checkpoint blockade in the present invention, and other immunotherapy methods may have similar effects.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention. In particular, as long as there is no structural conflict, the various technical features mentioned in each embodiment can be combined in any way. The present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions that fall within the scope of the claims.

Claims

1. A 125 I nanometer implant particle, characterized in that, Said 125 The nanometer implanted particle has a core-shell structure, which includes an implanted particle core, and a 125 I, and coated on the implanted particle core and the 125 I external covalent organic framework shell; the covalent organic framework shell is a covalent organic framework shell containing type I photosensitizer.

2. The method according to claim 1 125 I nanometer implant particle, characterized in that, The covalent organic framework shell containing type I photosensitizer includes a porphyrin-containing organic framework shell; Preferably, the thickness of the covalent organic framework shell is 10 nm to 50 nm.

3. The method according to claim 1 125 I nanometer implanted particle, characterized in that, The core of the implanted particle is a core-shell structure, which includes a nano core particle and a silver shell covering the outside of the nano core particle; 125 I is adsorbed on the outer surface of the silver shell; Preferably, the radius of the nano core particle is 25 nm to 50 nm, and the thickness of the silver shell is 3 nm to 10 nm.

4. The method according to claim 1 125 I nanometer implant particle, characterized in that, The nano core particles include at least one of gold nanoparticles and heavy metal particles; Preferably, the heavy metal particles are selected from at least one of bismuth sulfide, hafnium oxide and nano-platinum.

5. A method according to any one of claims 1 to 4 125 A method for preparing nanometer implant particles, characterized in that: The following steps are involved: preparing implantable particle cores; The implanted particle core after the first surface modification is subjected to a covalent organic framework synthesis reaction to obtain an implanted particle core@covalent organic framework shell; After the implanted particle core@covalent organic framework shell is subjected to a second surface modification, the implanted particle core@covalent organic framework shell is placed in a 125 I solution was allowed to stand, and the obtained solution was centrifuged and washed. 125 I nanometer implant particles.

6. The preparation method according to claim 5, characterized in that: The said 125 In the solution of I 125 I activity is 50μCi~500μCi.

7. The preparation method according to claim 5, characterized in that: The implanted particle core comprises an Au@Ag core, Under acidic conditions and inert gas protection, the Au@Ag core after the first surface modification is reacted with 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin and 2,5-dihydroxyterephthalaldehyde at 120° C. in the dark for 24 h to 72 h to obtain Au@Ag@TAPP-COF.

8. The preparation method according to claim 5 or 7, characterized in that: The second surface modification uses a solution containing polyethylene glycol; the first surface modification uses a solution containing polyvinyl pyrrolidone.

9. The preparation method according to claim 7, characterized in that: The preparation of the Au@Ag core includes: The Au@Ag core particles are mixed with a solution containing polyethyleneimine and a solution containing ascorbic acid, and a solution containing silver ions is added dropwise until the color of the solution changes to orange-red, thereby obtaining the Au@Ag core.

10. The method according to any one of claims 1 to 4 125 I nanometer implanted particles or prepared by the preparation method according to any one of claims 5-9 125 Application of nanoparticle implants in brachytherapy.

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