Preparation method and application of anti-tumor nanoparticles based on lactate oxidase and Prussian blue

Abstract

The invention discloses a preparation method and application of anti-tumor nano-particles based on lactate oxidase and Prussian blue. The nano-particles are hollow Prussian blue nano-particles (PBNPs), the surfaces of the hollow Prussian blue nano-particles (PBNPs) are modified with CXCR1 targeting polypeptide, and the surfaces of the hollow Prussian blue nano-particles (PBNPs) are internally loaded with lactate oxidase (LOD); the amino acid sequence of the CXCR1 targeting polypeptide is as follows: SAKELRCQCIKTYSKPFHPK, and the amino acid sequence of the CXCR1 targeting polypeptide is as follows: Comprising the following steps: S1, synthesizing through a polypeptide synthesizer, and grafting hydroxyl in the rhodamine fluorescent dye to amino of initial serine for tracing. By grafting the CXCR1 targeting polypeptide and loading the LOD catalytic enzyme, the nano catalysis-photo-thermal material (CXCR1-LOD at PBNPs) with targeting activity and biological enzyme activity can be successfully prepared. Through further in-vivo and in-vitro experimental studies, the anti-tumor effect and the molecular mechanism of the CXCR1-LOD coated PBNPs nano material can be fully confirmed. The material combines targeting property, catalytic property and photo-thermal property, and is a further improvement on early-stage research, so that a new strategy and a new method are provided for treatment of osteosarcoma.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method and application of an anti-tumor nanoparticle based on lactic acid oxidase and Prussian blue. BACKGROUND

[0002] Osteosarcoma is a primary malignant bone tumor with high invasiveness and destructiveness, mainly occurring in children and adolescents, with an incidence of more than 20% of all primary malignant bone tumors. It has the characteristics of high malignancy and easy distant metastasis, which seriously threatens the life and health of patients. It is urgent to take active and effective prevention and control measures to curb the development of osteosarcoma, so as to maximize the quality of life of patients.

[0003] Lactic acid metabolism is one of the important pathways of energy metabolism of tumor cells, and is closely related to the growth, invasion and metastasis of tumors. Osteosarcoma cells proliferate rapidly and have high energy demand. Due to uneven distribution of blood vessels and abnormal structure inside the tumor, oxygen supply is often insufficient, so it mainly relies on glycolysis to produce lactic acid to obtain energy. Even under sufficient oxygen conditions, osteosarcoma cells will perform glycolysis (i.e. aerobic glycolysis or Warburg effect) to continuously generate lactic acid to meet the energy demand of rapid growth and division. This process not only provides energy for tumor cells, but also promotes the malignant progression of tumors by changing the tumor microenvironment. Lactic acid accumulation can cause the extracellular fluid of tumor cells to be acidic, and this acidic environment is conducive to tumor invasion and metastasis, and can enhance the resistance of tumor cells to chemotherapy drugs. In summary, lactic acid metabolism plays a crucial role in the occurrence, development and prognosis of osteosarcoma, not only can provide strong support for the precise diagnosis of osteosarcoma, but also can help to develop new treatment targets and strategies, and bring hope for improving the treatment effect and quality of life of osteosarcoma patients.

[0004] In the applicant's previous research, a kind of nanoparticle was developed targeting the Warburg effect. Glucose oxidase (GOD) selectively catalyzes the glucose inside the tumor to generate hydrogen peroxide (H2O2) and gluconic acid. H2O2 can be catalyzed by Fe3O4 in the system to generate highly toxic active oxygen species hydroxyl radical (·OH) under acidic conditions, which can induce tumor cell apoptosis. Another study has also found that lactic acid oxidase (LOD) can catalyze lactic acid to generate H2O2 and pyruvic acid. The applicant's previous research has also designed a polypeptide fragment that can target CXCR1, achieving molecular targeted diagnosis and treatment of osteosarcoma. Therefore, LOD can accelerate the consumption of lactic acid in the tumor microenvironment, thereby inhibiting the progression of tumors, and the CXCR1-targeting polypeptide can achieve targeting of osteosarcoma, and the combination of the two can further improve the treatment effect of osteosarcoma.

[0005] In the applicant's previous authorized patent application (patent number: ZL202310049810.5, 2024.12.17), Prussian blue nanoparticles (PB nanoparticles, PBNPs) were used as the main component. By inhibiting the CXCR1 signaling pathway through CXCR1-targeting peptides, the photothermal effect of PBNPs was enhanced, thereby improving the therapeutic effect of osteosarcoma. However, this nanoparticle only increased the anti-tumor effect by inhibiting the signaling pathway. Studies have shown that PBNPs can also catalyze the generation of ·OH from H2O2, promoting tumor cell apoptosis; this is the biocatalytic function of PBNPs. Lactic acid can be decomposed into H2O2 under the catalysis of LOD. Therefore, if LOD can be loaded onto PBNPs, it can continuously provide H2O2 generated from lactic acid decomposition to induce osteosarcoma cell apoptosis. Furthermore, combined with the photothermal effect of PBNPs, this patent application will further improve the therapeutic effect of osteosarcoma.

[0006] In summary, this patent application aims to develop a more effective targeted therapeutic agent for osteosarcoma based on lactate oxidase, CXCR1-targeting peptides, and Prussian blue. This patent will use osteosarcoma as the research subject to verify the antitumor effect of LOD-modified CXCR1-targeting Prussian blue nanoparticles.

[0007] No effective solutions have yet been proposed to address the problems in the relevant technologies. Summary of the Invention

[0008] In view of the problems in related technologies, this invention proposes a method for preparing and applying anti-tumor nanoparticles based on lactate oxidase and Prussian blue, so as to overcome the above-mentioned technical problems existing in the existing related technologies.

[0009] Therefore, the specific technical solution adopted by the present invention is as follows:

[0010] A method for preparing and applying antitumor nanoparticles based on lactate oxidase and Prussian blue, wherein the nanoparticles are hollow Prussian blue nanoparticles (PBNPs) with CXCR1 targeting peptides modified on the surface and LOD oxidase loaded inside.

[0011] The amino acid sequence of the CXCR1 targeting peptide is: SAKELRCQCIKTYSKPFHPK;

[0012] Includes the following steps:

[0013] S1. After synthesis using a peptide synthesizer, the hydroxyl groups in the rhodamine fluorescent dye are grafted onto the amino group of the starting serine for tracer purposes.

[0014] Preparation of S2 and CXCR1-LOD@PBNPs particles: Hollow mesoporous PBNPs were prepared by surface protection / internal selective etching. First, polyvinylpyrrolidone (PVP) (3.0 g), K3[Fe(CN)6] (132 mg), and HCl solution (0.01 M, 40 mL) were mixed under magnetic stirring. After obtaining a transparent solution, the solution was placed in an 80℃ electric oven for 24 hours, and then washed repeatedly by centrifugation with distilled water to obtain mesoporous PBNPs. Subsequently, PVP (5 mg / mL) was added to the solution of mesoporous PBNPs (1 mg / mL), and the mixture was stirred in a Teflon container. After 3 hours, the solution was placed in a stainless steel pressure cooker at 140℃ and heated in an electric oven for 4 hours. After centrifugation, the precipitate was washed several times with distilled water and freeze-dried to obtain hollow mesoporous PBNPs.

[0015] S3. Loading LOD onto hollow mesoporous PBNPs: Take 4 ml of LOD solution (10 mg / mL) and add 10 mg of lyophilized hollow mesoporous PBNPs. Stir at room temperature; centrifuge to remove unloaded free drug, wash twice with PBS, lyophilize, collect the unloaded LOD supernatant, determine the content of remaining oxidase by colorimetry, and calculate the loading amount.

[0016] S4. Modification of the fluorescent CXCR1 targeting peptide: Since PBNPs are negatively charged, positively charged NH2-PEG-NH2 is adsorbed onto the surface of the nanoparticles synthesized in step S3 using a charge adsorption method, thereby amylating the nanoparticle surface. The fluorescent CXCR1 targeting peptide is grafted onto the amylated nanoparticles using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) activation method. Finally, the amylated nanoparticles are grafted with the carboxyl group of glutamic acid in the molecular probe, and then freeze-dried and stored for later use.

[0017] Ultimately, three types of nanoparticles were obtained: CXCR1-LOD@PBNPs, LOD@PBNPs, and PBNPs.

[0018] Preferably, in step S2, the intermediate product is LOD@PBNPs, which is obtained by repeated centrifugation and washing, and stored at 4°C for later use.

[0019] Preferably, in step S4, the freeze dryer freezes the product and then stores it at 4°C for later use.

[0020] According to another aspect of the invention, the use of antitumor nanoparticles based on lactate oxidase and Prussian blue in the preparation of a drug that inhibits CXCR1 activity is provided.

[0021] According to another aspect of the invention, the use of antitumor nanoparticles based on lactate oxidase and Prussian blue in the preparation of a medicament for inhibiting osteosarcoma is provided.

[0022] The beneficial effects of this invention are as follows: Based on previous research, using PBNPs as the main body, and by grafting CXCR1-targeting peptides and loading LOD catalytic enzymes, a nanocatalytic-photothermal material (CXCR1-LOD@PBNPs) with both targeting and bioenzymatic activity can be successfully prepared. Further in vivo and in vitro experimental studies can fully confirm the antitumor effect and molecular mechanism of CXCR1-LOD@PBNPs nanomaterials. This material combines targeting, catalysis, and photothermal properties, and represents a further improvement on previous research, which will provide new strategies and methods for the treatment of osteosarcoma. Compared with previously synthesized CXCR1@PBNPs nanoparticles, it exhibits better therapeutic effects against osteosarcoma. This is crucial for developing effective methods to control osteosarcoma recurrence and metastasis, locating tumor sites, improving the treatment effect of osteosarcoma, and reducing systemic adverse reactions caused by chemotherapy drugs. The expected results have significant theoretical and applied value. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a diagram illustrating the nanoparticle preparation process according to an embodiment of the present invention;

[0025] Figure 2 This is a transmission electron microscope image of CXCR1-LOD@PBNPs nanoparticles according to an embodiment of the present invention;

[0026] Figure 3 This refers to the photothermal effect of CXCR1-LOD@PBNPs nano-targeting particles according to an embodiment of the present invention;

[0027] Figure 4 This is a diagram illustrating the in vivo targeting and anti-osteosarcoma effects of CXCR1-LOD@PBNPs nanoparticles according to an embodiment of the present invention.

[0028] Figure 5 Immunohistochemical staining according to an embodiment of the present invention shows that CXCR1 and HIF-1α were significantly inhibited in the CXCR1-LOD / PBNPs treatment group. Detailed Implementation

[0029] To further illustrate the various embodiments, the present invention provides accompanying drawings, which are part of the disclosure of the present invention. These drawings are mainly used to illustrate the embodiments and can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. With reference to these drawings, those skilled in the art should be able to understand other possible implementation methods and the advantages of the present invention. The components in the drawings are not drawn to scale, and similar component symbols are generally used to represent similar components.

[0030] According to embodiments of the present invention, a method for preparing and applying antitumor nanoparticles based on lactate oxidase and Prussian blue is provided.

[0031] Example 1:

[0032] Synthesis of CXCR1-LOD@PBNPs nanoparticles.

[0033] like Figure 1 As shown, according to an embodiment of the present invention, the preparation method and application of antitumor nanoparticles based on lactate oxidase and Prussian blue are based on hollow Prussian blue nanoparticles (PBNPs) with CXCR1 targeting peptides modified on the surface and LOD oxidase loaded inside.

[0034] The amino acid sequence of the CXCR1 targeting peptide is: SAKELRCQCIKTYSKPFHPK;

[0035] Includes the following steps:

[0036] S1. After synthesis using a peptide synthesizer, the hydroxyl groups in the rhodamine fluorescent dye are grafted onto the amino group of the starting serine for tracer purposes.

[0037] Preparation of S2 and CXCR1-LOD@PBNPs particles: Hollow mesoporous PBNPs were prepared by surface protection / internal selective etching. First, polyvinylpyrrolidone (PVP) (3.0 g), K3[Fe(CN)6] (132 mg), and HCl solution (0.01 M, 40 mL) were mixed under magnetic stirring. After obtaining a transparent solution, the solution was placed in an 80℃ electric oven for 24 hours, and then washed repeatedly by centrifugation with distilled water to obtain mesoporous PBNPs. Subsequently, PVP (5 mg / mL) was added to the solution of mesoporous PBNPs (1 mg / mL), and the mixture was stirred in a Teflon container. After 3 hours, the solution was placed in a stainless steel pressure cooker at 140℃ and heated in an electric oven for 4 hours. After centrifugation, the precipitate was washed several times with distilled water and freeze-dried to obtain hollow mesoporous PBNPs.

[0038] S3. Loading LOD onto hollow mesoporous PBNPs: Take 4 ml of LOD solution (10 mg / mL) and add 10 mg of lyophilized hollow mesoporous PBNPs. Stir at room temperature; centrifuge to remove unloaded free drug, wash twice with PBS, lyophilize, collect the unloaded LOD supernatant, determine the content of remaining oxidase by colorimetry, and calculate the loading amount.

[0039] S4. Modification of the fluorescent CXCR1 targeting peptide: Since PBNPs are negatively charged, positively charged NH2-PEG-NH2 is adsorbed onto the surface of the nanoparticles synthesized in step S3 using a charge adsorption method, thereby amylating the nanoparticle surface. The fluorescent CXCR1 targeting peptide is grafted onto the amylated nanoparticles using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) activation method. Finally, the amylated nanoparticles are grafted with the carboxyl group of glutamic acid in the molecular probe, and then freeze-dried and stored for later use.

[0040] Ultimately, three types of nanoparticles were obtained: CXCR1-LOD@PBNPs, LOD@PBNPs, and PBNPs.

[0041] Previous studies may have explored LOD bioenzymes and CXCR1-targeting peptides separately, but did not focus on systematically verifying their synergistic effect. Since both CXCR1-targeting peptides and LOD enzymes are highly targeted, their combination can more precisely target lactate metabolism in tumor cells, reduce non-specific effects on normal cells, and improve the efficacy and safety of treatment. This represents a further improvement and enhancement of the previously filed patent. Furthermore, combining the biocatalytic and photothermal effects of PBNPs can further enhance the anti-tumor activity of nanoparticles. This approach is significantly innovative, and the research process is systematic and highly targeted, providing a new perspective and method for tumor metabolism-related research. In summary, this patent combines LOD bioenzymes, CXCR1-targeting peptides, and Prussian blue, which not only achieves targeted action but also leverages the advantages of Prussian blue nanoparticles' biocatalytic and photothermal effects, greatly improving the treatment efficacy for osteosarcoma.

[0042] In step S2, the intermediate product is LOD@PBNPs, which is obtained by repeated centrifugation and washing, and stored at 4°C for later use.

[0043] In step S4, the freeze dryer dries the food and then stores it at 4°C for later use.

[0044] According to another aspect of the invention, the use of antitumor nanoparticles based on lactate oxidase and Prussian blue in the preparation of a drug that inhibits CXCR1 activity is provided.

[0045] According to another aspect of the invention, the use of antitumor nanoparticles based on lactate oxidase and Prussian blue in the preparation of a medicament for inhibiting osteosarcoma is provided.

[0046] Example 2:

[0047] Transmission electron microscopy (TEM) scan of CXCR1-LOD@PBNPs nanoparticles.

[0048] Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-2100F high-resolution transmission electron microscope (TEM) with an operating voltage of 200 kV. Before testing, the samples were ground into powder and ultrasonically dispersed in ethanol for 10 min. TEM sample preparation involved adding one drop of 1 mg / mL nanoparticles to a 230-mesh copper grid and drying at room temperature. Three different fields of view were randomly selected during scanning.

[0049] like Figure 2 The transmission electron microscope (TEM) image of the CXCR1-LOD@PBNPs nanoparticles shown indicates that we successfully synthesized CXCR1-LOD@PBNPs nanoparticles. The scanning electron microscope (SEM) shows that the nanoparticles are well dispersed and have uniform particle size, which meets the requirements for further experiments.

[0050] Example 3:

[0051] Verification of the photothermal targeted antitumor effect and investigation of the molecular mechanism of CXCR1-LOD@PBNPs nanoparticles.

[0052] 1. Establishment of an orthotopic animal model of osteosarcoma

[0053] Saos2-lung osteosarcoma cells were inoculated into the medullary cavity of the tibia of nude mice to establish an orthotopic osteosarcoma model. Osteosarcoma cells in the logarithmic growth phase were selected, and the cell concentration was adjusted to an appropriate range, typically 1 × 10⁻⁶. 5 / mL to 1×10 6 / mL. Nude mice were routinely anesthetized, and the prepared cell suspension was injected into the medullary cavity of the proximal tibia using a microsyringe. Care should be taken to control the injection rate and volume during the injection process to avoid cell extravasation or damage to surrounding tissues. Tumor growth was monitored in real-time using small animal in vivo imaging, recording tumor size, morphology, and metastasis.

[0054] 2. Tumor monitoring and data analysis

[0055] The treatment groups were as follows: control group, CXCR1-LOD@PBNPs+laser group, LOD@PBNPs+laser group, and PBNPs+laser group. After the set treatment, the tumor-bearing mice were continued to be fed, and changes in tumor volume and metastasis were observed using PAI, MRI, and small animal in vivo imaging. The observation was continued for 4 weeks. After the experiment, the survival rate and metastasis rate of the tumor-bearing nude mice in each group were statistically analyzed. Subsequently, histological sections and immunohistochemistry were used to detect and analyze the protein indicators related to the CXCR1 / HIF-1α signaling pathway.

[0056] 3. Experimental Results

[0057] After successfully establishing an orthotopic tibial osteosarcoma model in nude mice, we monitored temperature changes in tumor-bearing mice injected with nanoparticles under NIR 808nm illumination. Under 808nm illumination, the CXCR1-LOD / PBNPs nanoparticles in the tumor-bearing mice could raise the temperature to 50.5℃, while unmodified PBNPs could only raise it to approximately 43.8℃. (See attached image) Figure 3 This indicates that nanoparticles have a good photothermal effect.

[0058] Subsequently, in vivo imaging in small animals revealed strong enrichment (red fluorescence) of CXCR1-LOD / PBNPs nanoparticles at the tumor site using in vivo imaging. This indicates that CXCR1-LOD / PBNPs nanoparticles possess strong targeting capabilities. (See attached image) Figure 4 A). Finally, after 4 weeks of continuous observation in tumor-bearing nude mice, tumor volume was compared. The results showed that CXCR1-LOD / PBNPs had the best inhibitory effect on osteosarcoma growth (see appendix). Figure 4 B). The above results confirm that loading CXCR1-targeting peptides and lactate oxidase onto PBNPs can significantly improve the photothermal targeted therapy effect for osteosarcoma.

[0059] Finally, we investigated the anti-tumor mechanism of CXCR1-LOD@PBNPs nanoparticles. Immunohistochemical results showed that CXCR1 and HIF-1α were significantly inhibited in the CXCR1-LOD / PBNPs nanoparticle treatment group (see appendix). Figure 5 Therefore, the above results confirm that loading CXCR1-targeting peptides and lactate oxidase onto PBNPs can significantly improve the photothermal targeted therapy effect of osteosarcoma, and this effect is achieved by inhibiting the CXCR1 / HIF-1α signaling pathway.

[0060] In summary, utilizing the technical solution described above, and based on previous research, a nanocatalytic-photothermal material (CXCR1-LOD@PBNPs) with both targeting and bioenzymatic activity can be successfully prepared by grafting CXCR1-targeting peptides and loading LOD catalytic enzymes onto PBNPs. Further in vivo and in vitro experiments have fully demonstrated the antitumor effects and molecular mechanisms of the CXCR1-LOD@PBNPs nanomaterials. This material combines targeting, catalytic, and photothermal properties, and represents a further improvement upon previous research, providing new strategies and methods for the treatment of osteosarcoma. Compared to previously synthesized CXCR1@PBNPs nanoparticles, it exhibits better therapeutic effects against osteosarcoma. This is crucial for developing effective methods to control osteosarcoma recurrence and metastasis, locating tumors, improving the efficacy of osteosarcoma treatment, and reducing systemic adverse reactions caused by chemotherapy drugs. The expected results have significant theoretical and applied value.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing antitumor nanoparticles based on lactate oxidase and Prussian blue, characterized in that, Based on the fact that the nanoparticles are hollow Prussian blue nanoparticles with CXCR1 targeting peptides modified on the surface and LOD catalytic enzymes loaded inside. The amino acid sequence of the CXCR1 targeting peptide is: SAKELRCQCIKTYSKPFHPK; Includes the following steps: S1. After synthesis using a peptide synthesizer, the hydroxyl groups in the rhodamine fluorescent dye are grafted onto the amino group of the starting serine for tracer purposes. Preparation of S2 and CXCR1-LOD@PBNPs particles: Hollow mesoporous PBNPs were prepared by surface protection / internal selective etching. First, polyvinylpyrrolidone, K3[Fe(CN)6], and HCl solution were mixed under magnetic stirring. After obtaining a transparent solution, the solution was placed in an 80℃ electric oven for 24 hours, and then washed repeatedly by centrifugation with distilled water to obtain mesoporous PBNPs. Subsequently, PVP was added to the mesoporous PBNPs solution and stirred in a Teflon container. After 3 hours, the solution was placed in a stainless steel pressure cooker at 140℃ and heated in an electric oven for 4 hours. After centrifugation, the precipitate was washed several times with distilled water and freeze-dried to obtain hollow mesoporous PBNPs. S3. Loading LOD onto hollow mesoporous PBNPs: Take 4 ml of LOD solution and add 10 mg of lyophilized hollow mesoporous PBNPs, stir at room temperature; centrifuge to remove unloaded free drug, wash twice with PBS, lyophilize, collect the unloaded LOD supernatant, determine the content of remaining oxidase by colorimetry, and calculate the loading amount. S4. Modification of the fluorescent CXCR1 targeting peptide: Since PBNPs are negatively charged, positively charged NH2-PEG-NH2 is adsorbed onto the surface of the nanoparticles synthesized in step S3 using a charge adsorption method, thereby amylating the nanoparticle surface. The fluorescent CXCR1 targeting peptide is grafted onto the amylated nanoparticles using a 1-ethyl-3-carbodiimide (EDC) activation method. Finally, the amylated nanoparticles are grafted with the carboxyl group of glutamic acid in the molecular probe, and then freeze-dried and stored for later use. Ultimately, three types of nanoparticles were obtained: CXCR1-LOD@PBNPs, LOD@PBNPs, and PBNPs.

2. The method for preparing antitumor nanoparticles based on lactate oxidase and Prussian blue according to claim 1, characterized in that, In step S2, the intermediate product is LOD@PBNPs, which is obtained by repeated centrifugation and washing, and stored at 4°C for later use.

3. The method for preparing antitumor nanoparticles based on lactate oxidase and Prussian blue according to claim 1, characterized in that, In step S4, the freeze dryer dries the food and then stores it at 4°C for later use.

4. Use of the antitumor nanoparticles based on lactate oxidase and Prussian blue according to any one of claims 1-3 in the preparation of a drug that inhibits CXCR1 activity.

5. Use of the antitumor nanoparticles based on lactate oxidase and Prussian blue according to any one of claims 1-3 in the preparation of a drug for inhibiting osteosarcoma.

Images