Preparation method of positive and negative CT (Computed Tomography) nano contrast agent and application of positive and negative CT nano contrast agent in detecting hydrogen peroxide microenvironment of diseases

Abstract

According to the preparation method of the positive and negative CT nano contrast agent and the application of the positive and negative CT nano contrast agent in a hydrogen peroxide microenvironment for detecting diseases, KMnO4 is used as a MnO2 raw material, BSA is used as a modifier, the obtained MnO2-coated BSA nano contrast agent serves as the positive and negative CT nano contrast agent, and the positive and negative CT nano contrast agent is low in cost, easy to obtain, mild in reaction condition, few in synthesis steps and simple in product treatment. Meanwhile, the positive and negative CT nano contrast agent prepared by the invention can realize rapid and sensitive CT image detection on a hydrogen peroxide microenvironment of a disease, and a detection signal is stable and is not interfered by external factors; realization results show that the prepared positive and negative CT nano contrast agent can realize accurate CT image detection of hydrogen peroxide expression of focuses of subcutaneous tumor model mice and muscle inflammation model mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of CT detection contrast technology, and relates to a CT contrast agent for detecting the hydrogen peroxide microenvironment, in particular to a preparation method of a positive and negative CT nano-contrast agent and its application in detecting the hydrogen peroxide microenvironment of diseases. Background Art

[0002] Hydrogen peroxide is the most common reactive oxygen molecule in organisms, and is significantly highly expressed in various diseases such as tumors and inflammation. Past studies have shown that the high expression of hydrogen peroxide significantly promotes tumor progression and metastasis, and its expression level is also of great significance for the occurrence and development of inflammatory diseases. Therefore, achieving sensitive imaging detection of hydrogen peroxide in the disease microenvironment is of great significance for monitoring the progression of tumor / inflammatory diseases and promoting the efficient treatment of diseases. CT imaging has the advantages of convenient operation, high spatial and temporal resolution, etc., and plays a very important role in the screening of clinical diseases including tumors. However, the soft tissue resolution of CT is not high, which results in its inability to achieve accurate detection of in-vivo metabolites and chemical substances. This is mainly because the attenuation of X-rays depends on the density of the substance itself. The presence and content of hydrogen peroxide in the local lesion tissue do not affect the tissue density of the diseased organ, resulting in the inability of CT imaging technology to achieve sensitive imaging of hydrogen peroxide in the lesion. Therefore, there is an urgent need to develop a suitable CT molecular contrast agent to achieve accurate detection of the disease hydrogen peroxide microenvironment.

[0003] Currently, most CT contrast agents (including iodine agents commonly used clinically) show high density and are generally defined as CT positive contrast agents. CT positive nano-contrast agents have many advantages in detecting tumors, such as multifunctional designability, enhanced permeability and retention (EPR) effect, etc. However, CT nano-positive contrast agents still cannot achieve in-vivo targeted detection of hydrogen peroxide, mainly because the uptake of CT nano-contrast agents lacks tissue specificity and metabolite response specificity. Tumors are significantly highly expressed in hydrogen peroxide compared with normal tissues, but this similar uptake will result in the inability to achieve sensitive imaging of hydrogen peroxide. Recently, Meng et al. innovatively developed a CT nano-negative contrast agent for detecting osteosarcoma. This contrast agent can specifically respond to the acidic microenvironment of osteosarcoma and produce hydrogen. The CT negative signal generated by hydrogen forms a strong contrast with the CT positive signal generated by the surrounding bones, thus achieving accurate CT diagnosis of osteosarcoma. Therefore, if a CT nano-positive contrast agent can be combined with a nano-negative contrast agent and specifically enriched in the site with high expression of hydrogen peroxide in the tumor or inflammation region to form a positive and negative CT signal contrast, it is expected to achieve sensitive CT diagnosis of the disease hydrogen peroxide microenvironment. Summary of the Invention

[0004] The present invention is directed to the above problems and provides a method for preparing a positive and negative CT nano-contrast agent for detecting the hydrogen peroxide microenvironment of diseases. MnO2@BSA nanosheets of manganese dioxide (MnO2) modified with bovine serum albumin (BSA) were designed and synthesized as CT imaging nano-contrast agents. After enrichment in the lesion area, the nano-contrast agent initially exhibits a positive CT signal; over time, the MnO2@BSA contrast agent in the tumor / inflammatory site specifically responds to the hydrogen peroxide microenvironment in the local lesion, releases oxygen and causes the CT value in the lesion area to gradually decrease, resulting in a negative contrast of gas. Therefore, the CT signal value in the tumor / inflammatory area decreases significantly, forming a strong contrast between the CT negative signal and the positive signal, thereby realizing sensitive CT detection of the hydrogen peroxide microenvironment of diseases.

[0005] To achieve the above object, the technical solutions provided by the present invention are as follows:

[0006] In the first aspect of the present invention, a method for preparing a positive and negative CT nano-contrast agent is provided, which is outlined as follows: Using KMnO4 as the raw material for MnO2 and BSA as the modifier, the MnO2@BSA nano-contrast agent is obtained as the positive and negative CT nano-contrast agent.

[0007] Preferably, the specific preparation steps of the contrast agent are as follows: Add the KMnO4 solution to the BSA solution under vigorous stirring, where the molar ratio between BSA and KMnO4 is 18-20:1; after stirring at 37 °C for at least 2 h, dialyze with ultrapure water for 24 hours and freeze-dry to synthesize the MnO2@BSA nano-contrast agent.

[0008] KMnO4 is reduced to MnO2 (brownish-black precipitate) in a neutral / weakly alkaline medium, and then BSA is adsorbed on the surface of the MnO2 nanoparticles through electrostatic interaction, hydrophobic interaction or hydrogen bond to form the MnO2@BSA nano-contrast agent:

[0009] MnO 4- + 2H2O + 3e - → MnO2 + 4OH - .

[0010] Furthermore, both BSA and KMnO4 are first prepared into solutions and then mixed. The concentration of the BSA solution is preferably 35-36 mg / mL, and the concentration of the KMnO4 solution is preferably 10-11 mg / mL; the stirring condition is 500-800 r / min, and the stirring time is 2 h.

[0011] Furthermore, the freeze-drying condition is -50 °C, 1-30 Pa.

[0012] In the second aspect of the present invention, a positive and negative CT nano-contrast agent prepared by the above method is provided. The hydrodynamic diameter of the contrast agent at pH = 7.4 is concentrated around 10 nm.

[0013] In the third aspect of the present invention, an application of the above positive and negative CT nano-contrast agent in the preparation of a reagent for detecting the microenvironment of hydrogen peroxide in diseases is provided. The positive and negative CT nano-contrast agent can generate positive and negative CT density signals in a hydrogen peroxide solution environment.

[0014] Preferably, the microenvironment of hydrogen peroxide in diseases refers to the lesion areas with high expression of hydrogen peroxide in tumors or inflammations, such as pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), lung cancer (non-small cell lung cancer, NSCLC), breast cancer (triple-negative subtype, TNBC), glioblastoma (GBM), liver cancer (HCC), melanoma, prostate cancer, ovarian cancer, etc.

[0015] Inflammation with high expression of hydrogen peroxide (H2O2) is a common feature of various acute and chronic inflammatory diseases, infections, and tissue injuries. H2O2 is both an effector molecule and a regulatory signaling molecule of the inflammatory response, and its abnormal level is closely related to disease progression. Hydrogen peroxide (H2O2) is highly expressed in various inflammations, such as infectious inflammations: bacterial infections (such as sepsis), chronic viral infections (such as HIV, HBV); autoimmune diseases: rheumatoid arthritis (RA), inflammatory bowel disease (IBD); metabolic inflammations: diabetic complications, atherosclerosis; neuroinflammations: Alzheimer's disease (AD) and Parkinson's disease (PD); skin inflammations: psoriasis and contact dermatitis, etc.

[0016] Experimental results show that the positive and negative CT nano-contrast agent MnO2@BSA prepared by the above method can respond to the hydrogen peroxide microenvironment in tumor / inflammation lesion areas in subcutaneous tumor model mice and muscle inflammation model mice, realizing the application of detecting hydrogen peroxide in diseases.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) The present invention uses BSA and KMnO4 as the main raw materials, which are cheap and easily available, the reaction conditions are mild, the synthesis steps are few, and the product treatment is simple;

[0019] (2) The positive and negative CT nano-contrast agent prepared by the present invention can achieve rapid and sensitive CT imaging detection of the microenvironment of hydrogen peroxide in diseases, the detection signal is stable, and it is not interfered by external factors; the experimental results show that the prepared positive and negative CT nano-contrast agent can achieve accurate CT imaging detection of the hydrogen peroxide expression in the lesions of subcutaneous tumor model mice and muscle inflammation model mice. Description of the Drawings

[0020] Figure 1Shows the transmission electron microscopy (TEM) image of the positive and negative CT nano-contrast agent MnO2@BSA prepared in Example 1.

[0021] Figure 2 Shows the hydrodynamic diameter of the positive and negative CT nano-contrast agent MnO2@BSA prepared in Example 1 at pH = 7.4.

[0022] Figure 3 Shows the CT images of the positive and negative CT nano-contrast agent MnO2@BSA in Example 2 in aqueous solutions with different hydrogen peroxide concentrations (C H2O2 = 0.1 / 1 / 5 / 10 / 20 / 40 mM).

[0023] Figure 4 Shows the CT images of the positive and negative CT nano-contrast agent MnO2@BSA in Example 3 for detecting local hydrogen peroxide in subcutaneous tumor-bearing mice.

[0024] Figure 5 Shows the CT images of the positive and negative CT nano-contrast agent MnO2@BSA in Example 3 for detecting local hydrogen peroxide in a muscle inflammation model. Detailed implementation methods

[0025] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of traditional methods, such methods being well known to those of ordinary skill in the art and being described in many publications.

[0026] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention, and the preferred implementation methods and materials described in the detailed implementation methods are for illustrative purposes only.

[0027] Example 1: Preparation and characterization of a novel positive and negative CT nano-contrast agent MnO2@BSA

[0028] 1. Preparation of MnO2@BSA nanosheets

[0029] Dissolve 250 mg of BSA in 7 mL of ultrapure water; add 31.6 mg of KMnO4 dissolved in 3 mL of ultrapure water under vigorous stirring; magnetically stir at 37 °C for 2 hours, dialyze with ultrapure water for 24 hours and lyophilize to synthesize the MnO2@BSA nano-contrast agent.

[0030] All the reagents used in the above steps were directly purchased from reagent companies, and all reagents were not purified in any way except as otherwise specified.

[0031] 2. Characterization of MnO2@BSA Nanosheets

[0032] Perform TEM characterization tests on the novel positive and negative CT nano-contrast agents obtained in the above steps; similarly, use dynamic light scattering (DLS) to characterize the hydrodynamic diameter of the positive and negative CT nano-contrast agents at pH = 7.4. The results show that the obtained MnO2 nano-contrast agents are in a monodisperse state with good dispersibility, and the particle size is about 9.7 nm ( Figure 1 ); the dynamic light scattering results indicate that the hydrodynamic diameter of the probe is about 9.7 nm ( Figure 2 ).

[0033] Example 2: MnO2@BSA Can Generate Positive and Negative CT Density Signals in H2O2 Aqueous Solution

[0034] Place the MnO2@BSA nanosheets prepared in Example 1 at a concentration of 0.5 mg / mL in solutions (2 mL) with different hydrogen peroxide concentrations (C H2O2 = 0.1 / 1 / 5 / 10 / 20 / 40 mM) and redisperse. Use a dual-energy CT imaging machine (100 mAs, 70 / Sn150 kV) to perform CT imaging at different time points.

[0035] Figure 3 The CT images of the novel positive and negative CT nano-contrast agent MnO2@BSA in solutions with different hydrogen peroxide concentrations are shown. Initially, MnO2@BSA has obvious CT positive enhancement performance, and the image appears green; as time goes by, at the set hydrogen peroxide concentrations, the CT negative signal gradually increases, the CT value decreases, and black concave areas appear in the image. For hydrogen peroxide with a concentration of 40 mM, negative contrast density appears in the CT image 10 minutes after the reaction; in hydrogen peroxide with the lowest concentration of 5 mM, the CT positive signal of the MnO2@BSA solution gradually decreases with the increase of the reaction time, and the negative density range increases, indicating that the probe can sensitively respond to low-concentration hydrogen peroxide to produce CT density changes. This result shows that the MnO2@BSA nano-contrast agent can sensitively respond to the hydrogen peroxide microenvironment in aqueous solution to produce CT positive and negative signal contrasts.

[0036] Example 3: Detection of Hydrogen Peroxide Microenvironment in a Subcutaneous Tumor Model of Mouse Hepatocellular Carcinoma by MnO2@BSA Nanosheets

[0037] (1) After culturing Huh7 cells in DEME culture medium (9% fetal bovine serum, 1% double antibody) for 48 h, digest them with trypsin, discard the supernatant after centrifugation, and then disperse them evenly in the above culture medium. Use a syringe to aspirate 200 µL (about 1 million cells) and inject it into the anterior side of the right hind limb of nude mice.

[0038] (2) After about 7 - 10 d, when the tumor volume is greater than 100 mm 3 , in vivo CT imaging experiment of subcutaneous liver cancer tumor model was carried out: 100 µL of aqueous solution containing MnO2@BSA nanosheets (10000 ppm) was directly injected into the tumor of tumor-bearing mice, and CT imaging was performed before and after injection respectively; CT images at different time points (Pre, 15 min, 30 min, 60 min, 120 min, 240 min) were compared.

[0039] After 30 min of MnO2@BSA injection, positive CT enhancement was observed in the tumor tissue in the region of interest (red circle); over time (60 - 240 min), a black area appeared in the tumor tissue and gradually increased ( Figure 4 ), which was due to the release of oxygen by MnO2@BSA nanoparticles when they encountered hydrogen peroxide in the tumor microenvironment. More importantly, the significant contrast and relative position between the positive CT signal region and the negative signal region were conducive to the sensitive detection of tumors.

[0040] Example 4: Detection of hydrogen peroxide microenvironment in mouse muscle inflammation model by MnO2@BSA nanosheets

[0041] Construction of mouse muscle inflammation model: Use a syringe to aspirate lipopolysaccharide (LPS) and inject it into the thigh muscle of mice. After about 1 d, in vivo CT imaging experiment of mouse muscle inflammation model was carried out: 100 µL of aqueous solution containing MnO2@BSA nano-contrast agent (10000 ppm) was locally injected into the left thigh muscle of the inflammation model mouse group, and CT imaging was performed before and after injection respectively; CT images at different time points (Pre, 15 min, 30 min, 60 min, 120 min, 240 min) were compared.

[0042] Figure 5 Shows the CT scan imaging of positive and negative CT nano-contrast agent MnO2@BSA for detecting hydrogen peroxide microenvironment in mouse muscle inflammation model. It can be found from the cross-sectional CT imaging pictures of mice that 15 minutes after injecting MnO2@BSA into the muscle tissue of the left leg of mice, the CT value enhancement image of the muscle tissue presented as orange-red. Over time, the positive CT signal gradually weakened, and the negative CT signal (black area) began to appear and became more around the positive CT contrast signal. This was because the nanoparticles reacted with hydrogen peroxide in the muscle inflammation tissue and released oxygen, and negative CT imaging contrast was generated during this process. It was more conducive to judging the production of hydrogen peroxide in the inflammatory tissue. In contrast, there was no obvious change in CT density in the contralateral muscle tissue of mice, indicating the excellent detection effect of MnO2@BSA nanoprobe.

[0043] The parts not described in the present invention are the same as those in the prior art or are implemented by using the prior art. The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A preparation method of positive and negative CT nano-contrast agents, characterized in that, Using KMnO4 as the raw material for MnO2 and BSA as the modifier, MnO2@BSA nanocontrast agent is obtained as a positive and negative CT nanocontrast agent.

2. The preparation method according to claim 1, characterized in that, The preparation steps of the contrast agent are as follows: Add the KMnO4 solution to the BSA solution under vigorous stirring. After stirring at 37 °C for a certain time, dialyze with ultrapure water for 24 hours and then lyophilize to synthesize the MnO2@BSA nanocontrast agent; among them, the molar ratio between BSA and KMnO4 is 18-20:

1.

3. The preparation method according to claim 2, characterized in that, The concentration of the BSA solution is 35-36 mg / mL, and the concentration of the KMnO4 solution is 10-11 mg / mL; the stirring condition is 500-800 r / min, and the stirring time is 2 h.

4. The preparation method according to claim 2, characterized in that, The lyophilization condition is -50 °C, 1-30 Pa.

5. A positive and negative CT nano-contrast agent, characterized in that, Prepared by the method according to any one of claims 1-4.

6. Use of the positive and negative CT nanocontrast agent according to claim 5 in the preparation of a reagent for detecting the hydrogen peroxide microenvironment of diseases.

7. The application according to claim 6, wherein The hydrogen peroxide microenvironment of the disease refers to the lesion area with high expression of hydrogen peroxide in tumors or inflammation.

8. The application according to claim 6, wherein The positive and negative CT nanocontrast agent can generate positive and negative CT density signals in a hydrogen peroxide solution environment.

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