PECAM-1 Targeted Diagnosis and Treatment Integrated Nanoprobe and Its Preparation Method and Application

Abstract

The present invention belongs to the technical field of nanoprobe, and specifically discloses a PECAM-1 targeted diagnosis and treatment integrated nanoprobe, its preparation method and application. It includes obtaining PECAM-1_4G6mAb: reacting hydrazinonicotinamide with PECAM-1_4G6 in an acidic environment, filtering the reaction product and reacting it with a radioactive nuclide washing solution under reducing agent conditions to obtain PECAM-1_4G6mAb; obtaining αPECAM-1_AT@HCNPs: adding a sufficient amount of N,N'-carbonylbis(succinimidyl) to an aqueous solution of HCNPs nanoparticles, adding PECAM-1_4G6mAb, and reacting at room temperature to obtain an aqueous solution of αPECAM-1-HCNPs nanoparticles. Then, the αPECAM-1-HCNPs nanoparticles and antithrombin are co-dissolved in equal proportions, and antithrombin is adsorbed and loaded onto the PECAM-1-HCNPs nanoparticles through electrostatic interaction, and purified to obtain αPECAM-1_AT@HCNPs. The present invention: uses a radionuclide-labeled single-chain antibody 4G6 with a half-life suitable for the CAR-T treatment time window (6-8 days) to capture early microvascular imaging of the CRS target organ.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoprobe, and particularly relates to a PECAM-1 targeted diagnosis and treatment integrated nanoprobe, a preparation method thereof and an application thereof. Background Art

[0002] The most serious adverse reaction of chimeric antigen receptor T cell immunotherapy (CAR-T) is cytokine release syndrome (CRS), with an incidence rate of about 77% - 85%. CRS is caused by the rapid proliferation of CAR-T cells after infusion in vivo and the excessive cascade release of cytokines (CK), resulting in high fever, hypotension, dyspnea, coagulation disorders, end-organ disorders, etc. in patients, and even death in severe cases. At present, the grading of CRS is mainly judged by clinical manifestations, lacking objective indicators; commonly used cytokine monitoring has late appearance of elevated indicators and difficult-to-quantify thresholds. Although existing studies have found some mechanisms leading to the occurrence of CRS in CAR-T therapy, no adaptive research has been carried out on this, and it has not been studied whether the monitoring and treatment of CRS can be achieved through corresponding targets. Therefore, exploring early molecular markers directly related to CRS and visualization techniques and establishing new means of monitoring and intervention will contribute to its clinical diagnosis and treatment. Summary of the Invention

[0003] Aiming at the above problems, the present invention provides a PECAM-1 targeted diagnosis and treatment integrated nanoprobe, a preparation method thereof and an application thereof, mainly to solve the problems that there is currently no good grading method for CRS and it is difficult to visually detect early molecular markers directly related to CRS.

[0004] To solve the above problems, the present invention adopts the following technical solutions:

[0005] The first aspect of the present invention relates to A preparation method of a PECAM-1 diagnosis and treatment integrated nanoprobe, comprising the following steps

[0006] S1. Obtain radioisotope-labeled PECAM-1_4G6mAb: React hydrazinonicotinamide with PECAM-1-4G6 in an acidic environment, filter the reaction product, and then react it with a radioisotope washing solution under reducing agent conditions to obtain the nuclide-labeled PECAM-1_4G6 mAb;

[0007] S2. Obtain αPECAM-1-AT@HCNPs: Add a sufficient amount of N,N'-carbonyldiimidazole to the aqueous solution of HCNPs nanoparticles, mix well, dialyze, add radionuclide-labeled PECAM-1_4G6mAb, and react at room temperature to obtain an aqueous solution of αPECAM-1_HCNPs nanoparticles. Then dissolve the αPECAM-1_HCNPs nanoparticles and antithrombin in equal proportions, and enable antithrombin to be adsorbed and loaded onto the PECAM-1_HCNPs nanoparticles through electrostatic interaction, and purify to obtain αPECAM-1_AT@HCNPs.

[0008] In some cases, the radionuclide is 177 Lu; the reducing agents are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stir and react at room temperature for 12 h.

[0009] In some cases, in step S2: Add 177 Lu-labeled PECAM-1_4G6 mAb, and obtain 177 an aqueous solution of Lu-αPECAM-1-HCNPs nanoparticles after stirring and reacting at room temperature. The amide reaction efficiency is relatively high at room temperature, and the coupling efficiency is high.

[0010] In some cases, in step S2, after antithrombin is electrostatically adsorbed and loaded onto antithrombin: perform ultrafiltration to remove unencapsulated and unloaded antithrombin to obtain purified αPECAM-1_AT@HCNPs.

[0011] In some cases, in step S2: Dissolve the αPECAM-1_HCNPs nanoparticles and antithrombin in equal proportions in ultrapure water, stir and react to enable antithrombin to be electrostatically loaded.

[0012] In some cases, the preparation method of the HCNPs nanoparticles is: Dissolve HES-CH in water, sonicate to obtain an aqueous solution of HES-CH, add chloroform dropwise to this solution while sonicating, rotary evaporate to remove chloroform, centrifuge the obtained aqueous solution of HCNPs, discard the precipitate and fully lyophilize to obtain HCNPs nanoparticle powder.

[0013] The second aspect of the present invention relates to the αPECAM-1_AT@HCNPs nanoparticles prepared by the foregoing method. Although the nanoparticles described in this article can be prepared by the foregoing method, they are not strictly limited to using the foregoing method, and the properties of the nanoparticles should be considered.

[0014] The third aspect of the present invention relates to the application of αPECAM-1_AT@HCNPs nanoparticles in the preparation of products for detecting or inhibiting the risk of CRS occurring in CAR-T therapy. Among them, CRS mainly occurs in chimeric antigen receptor T cell immunotherapy. Through the nanoparticles of the present invention, the detection of CRS can be achieved, which can better monitor the CAR-T treatment process and screen for risks. Moreover, the occurrence of CRS in chimeric antigen receptor T cell immunotherapy can also be inhibited by the nanoparticles.

[0015] The fourth aspect of the present invention relates to the application of αPECAM-1_AT@HCNPs nanoparticles in the preparation of imaging agents for CRS endothelial damage. Among them, αPECAM-1_AT@HCNPs nanoparticles can also monitor endothelial damage and the like that occur during CRS, and can more intuitively determine the location where endothelial damage occurs.

[0016] The fifth aspect of the present invention relates to the application of αPECAM-1_AT@HCNPs nanoparticles in the preparation of drugs for inhibiting CRS systemic inflammatory response diseases, wherein the CRS systemic inflammatory response diseases include vascular endothelial damage and vascular barrier dysfunction. Delivering related drugs and radionuclide probes with extracellular cleavage sites as targets helps to promote endothelial reconstruction and reduce CRS, and the present invention provides such a PECAM-1 diagnostic and therapeutic integrated nanoprobe that can achieve promoting endothelial reconstruction and reducing CRS.

[0017] The beneficial effects of the present invention are as follows: Using a radionuclide-labeled single-chain antibody 4G6 with a half-life suitable for the CAR-T treatment time window (6 - 8 days) to early capture the microvascular imaging of the CRS target organ; at the same time, guiding nanoparticles with good biocompatibility to load AT and precisely release it at the local site of CRS damage, inhibiting excessive coagulation activation and reducing inflammation. This strategy can early monitor and intervene in CRS and has great clinical transformation value. Brief Description of the Drawings

[0018] Figure 1 It is a CRS endothelial damage model related to CAR-T treatment.

[0019] Figure 2 It is for detecting the tumor burden of lymphoma mice by small animal in vivo imaging. (A) ELISA detection found that after administration of CAR-T, the levels of IL-1β (B) and IL-6 (C) in the peripheral blood of mice increased significantly; coagulation index detection revealed that APTT was prolonged during CRS and there was a statistical difference (D), while PT had no significant difference from the control group (E); immunohistochemistry showed that after treatment, a large number of CAR-T cells homed to the tumor and recruited a large number of macrophages (F); H&E staining maps of various organs in CRS model mice (G).

[0020] Figure 3 For 177 Characterization of Lu-αPECAM-1-AT@HCNPs nanoprobes. (A) TEM was used to observe the morphology of the nanoprobes; (B) DLS was used to detect the particle size distribution of the nanoprobes.

[0021] Figure 4 DIR was used as an alternative label for nanoparticles to detect 177 In vivo distribution of Lu-αPECAM-1-AT@HCNPs nanoprobes in a CRS mouse model. (A) Small animal in vivo imaging was used to detect the biodistribution of αPECAM-1-DIR@HCNPs in the heart, liver, spleen, lungs, and kidneys of CRS mice; (B) Quantitative analysis of the fluorescence intensity of αPECAM-1-DIR@HCNPs in each organ;

[0022] Figure 5 After 177 intervention with Lu-αPECAM-1-AT@HCNPs, the plasma inflammatory factor levels of the model mice in each group were detected;

[0023] Figure 6 To compare the leukocyte infiltration in organs such as the liver and lungs; to evaluate the effect of the intervention measures on the in vivo inflammatory response;

[0024] Figure 7 To evaluate the macromolecule leakage using immunofluorescence technology;

[0025] Figure 8 For 177 The research idea model involving Lu-αPECAM-1-AT@HCNPs nanoprobes. Specific implementation manners

[0026] The present invention will be further described below:

[0027] (I) Experimental methods

[0028] 1. Establish a CRS endothelial injury model related to CAR-T therapy

[0029] a. Prepare CD19 CAR-T cells: Collect healthy human peripheral blood by apheresis, isolate PBMCs, use magnetic beads to sort out CD4 + T cells and CD8 + T cells, add them to cell culture flasks pre-coated with CD3 and CD28 factors respectively, and stimulate them with IL-2 and IFN-γ to make them grow and divide faster. Transfect with lentivirus according to a certain MOI value, and wash away the virus after five days. Take a small amount of cells for cytokine release detection, killing detection, PCR detection, and flow cytometry detection of transfection rate to determine whether the CAR-T cell function is perfect;

[0030] b.Inducing CRS in mice after CAR-T infusion: Prepare several female SCID beige mice aged 6 - 8 weeks on D0. Inject 5×10 6 luciferase-labeled Raji cells into the abdominal cavity of each mouse. Perform in vivo fluorescence imaging on D3, D7, D10, and D14 to evaluate the tumor burden and exclude mice with extreme burden. On D14, inject 30×10 6 CAR-T into the abdominal cavity of each experimental group mouse, and inject an equal volume of normal saline into the control group. CRS can be detected in the experimental group mice on D16.

[0031] 2.Preparation and characterization of PECAM-1 theranostic nanoprobes

[0032] a.Prepare drug-loaded nanoparticles (HCNPs) by the classic Pickering emulsion solvent evaporation method. That is, dissolve 50 mg of HES-CH in 50 ml of deionized water, and use an ultrasonic crusher to sonicate for 10 min (frequency is 50 Hz, sonicate for 2 s, stop for 1 s) to obtain a 1 mg / ml HES-CH aqueous solution. Then slowly add 5 ml of chloroform to this solution while sonicating with an ultrasonic crusher for 5 min to obtain a milky white homogeneous oil / water mixed solution. Then use a rotary evaporator to fully evaporate the chloroform at 45 °C. After that, centrifuge the obtained HCNPs aqueous solution (5000 rpm, 10 min), discard the precipitate and fully lyophilize to obtain HCNPs nanoparticle powder. Similarly, prepare DIR@HCNPs nanoparticles according to the above method: after obtaining a 1 mg / ml HES-CH aqueous solution, slowly add 5 ml of a chloroform solution (1 mg / ml) of 1,1-dioctadecyl-3,3,3,3-tetramethyl-indotricarbocyanine iodide (DIR) to this solution, and the remaining steps are the same;

[0033] b.Radionuclide 177 Lu-labeled antibody: First, react hydrazinonicotinamide (HYNIC) with PECAM-1-4G6 in an acidic environment for 2 h; then filter the product and react it with fresh 177 Lu washing solution for 30 min. Take a small amount of the reaction product for radio thin layer chromatography to measure the labeling rate; use a PD-10 column for purification and measure the radiochemical purity by radio thin layer chromatography;

[0034] c. 177Preparation and Characterization of Lu-αPECAM-1-AT@HCNPs: At room temperature, sufficient N,N'-disuccinimidyl carbonate (DSC) was added to an aqueous solution of 1 mg / ml HCNPs and stirred overnight. Subsequently, dialysis was carried out for 24 h, and then 25 μg of radionuclide-labeled PECAM-1-4G6 mAb was added, and the reaction was stirred at room temperature for 3 h to finally obtain an aqueous solution of PECAM-1-HCNPs nanoparticles. Similarly, PECAM-1-DIR@HCNPs was prepared according to the above method (using a fluorescein-labeled probe and studying the in vivo distribution of the nanoprobe). Antithrombin (AT) and the above nanoparticles were dissolved in ultrapure water in equal proportions and stirred for 2 hours. AT was adsorbed and loaded onto the nanoparticles through electrostatic interaction. The obtained αPECAM-1-AT@HCNPs nanoparticles were ultrafiltered to remove the unloaded AT to obtain purified αPECAM-1-AT@HCNPs. The microscopic morphology of this nanoprobe was observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) (the results are as Figure 3 shown in).

[0035] 3. 177 Imaging Specificity of Lu-αPECAM-1-AT@HCNPs

[0036] 177 Detection of the Targeted Inflammatory Endothelial Imaging Function of Lu-αPECAM-1-AT@HCNPs in CRS Mice: 177 Lu-αPECAM-1-AT@HCNPs was injected into the tail vein of the model mice. SPECT was used to analyze the collected images, and the high-imaging sites were quantitatively analyzed to evaluate the high imaging sensitivity of the nanoprobe. After the SPECT examination, tissues of organs such as the brain, heart, liver, spleen, lung, and kidney were taken to detect the radionuclide amounts in each organ to understand their biodistribution.

[0037] 4. 177 Evaluation of the Endothelial Reconstruction and Anti-Inflammatory Functions of Lu-αPECAM-1-AT@HCNPs

[0038] a. After the CRS model mice were intervened with 177 Lu-αPECAM-1-AT@HCNPs, a hematology analyzer was used to detect the hematocrit of each group of model mice to measure the liquid leakage situation; fluorescently labeled low / high molecular weight dextran was infused into the tail vein, and immunofluorescence technology was used to evaluate the macromolecular leakage situation (the results are shown in Figure 7 Figure);

[0039] b. After the intervention with 177 Lu-αPECAM-1-AT@HCNPs, the plasma inflammatory factor levels of the above groups of model mice were detected (the results are as Figure 5As shown in the figure, the leukocyte infiltration in organs such as the liver, kidney, and lung was compared to evaluate the effect of the intervention measures on the inflammatory response in the body, such as Figure 6 shown 177 After the intervention of Lu-αPECAM-1-AT@HCNPs, it can effectively intervene and relieve the inflammatory response in the body;

[0040] c. Evaluation of the degree of tissue and organ inflammation: After 177 the intervention of Lu-αPECAM-1-AT@HCNPs, the mice were anesthetized and the liver, lung, and kidney were taken. The degree of inflammatory response in tissues and organs was observed by H&E staining, and the granulocyte infiltration in tissues and organs was observed by immunohistochemistry.

[0041] (II) Analysis: CAR-T cytokine storm (CRS) is an acute inflammatory syndrome caused by pro-inflammatory factors acting on the microvascular endothelium of organs such as the lung and liver. Its mechanism is related to the damage of the vascular barrier caused by CRS and the increase in plasma soluble PECAM-1 (sPECAM-1) due to the cleavage of vascular endothelial adhesion factor 1 (PECAM-1) on the surface of endothelial cells. Detecting the degree of damage of CRS to the endothelial system with the extracellular cleavage point of PECAM-1 as the target, whether early monitoring and intervention of CRS can be achieved is not clear. The present invention proposes to use PECAM-1 as the target, and by radiolabeling a monoclonal antibody (4G6) targeting PECAM-1IgD6 and modifying the AT-loaded nanoparticles, local imaging of CRS and inflammation inhibition are achieved, providing a new technology for the early monitoring and intervention of CRS. Such as Figure 8 In the present invention, by preparing a targeted PECAM-1 diagnosis and treatment integrated nanoprobe, a radionuclide 177 Lu with a suitable tracer half-life (6 - 8 days) for the CAR-T time window is used to label the antibody 4G6 that can target the IgD6 region on PECAM-1 to construct 177 nanoparticles (HCNPs) connected with Lu-αPECAM-1, and adsorb the macromolecular drug (antithrombin AT). 177 Lu-αPECAM-1-AT@HCNPs can achieve early capture and imaging of the target organs of CRS under the guidance of the radionuclide 177 Lu-labeled single-chain antibody 4G6; at the same time, the nanoparticles precisely release the loaded AT at the local site damaged by CRS, inhibiting excessive coagulation activation and reducing inflammation, which all prove the effectiveness of the nanoparticles involved in the present invention for the monitoring and treatment of CRS.

[0042] Those skilled in the art can clearly understand that various modifications can be made to the above embodiments without departing from the general spirit and concept of the present invention. All of them fall within the protection scope of the present invention. The protection scope of the present invention is subject to the appended claims of the present invention.

Claims

1. Preparation method of PECAM-1 targeted integrated diagnosis and treatment nanosensor, characterized in that, it includes the following steps: S1. Obtain radioisotope-labeled PECAM-1_4G6mAb: React hydrazinonicotinamide with PECAM-1-4G6 in an acidic environment, filter the reaction product, and react it with a radioisotope washing solution under reducing agent conditions to obtain a nuclide-labeled PECAM-1_4G6 mAb; S2. Obtain αPECAM-1-AT@HCNPs: Add a sufficient amount of N,N'-carbonyldisuccinimide to an aqueous solution of HCNPs nanoparticles, mix well, dialyze, add the nuclide-labeled PECAM-1_4G6mAb, and react at room temperature to obtain an aqueous solution of αPECAM-1-HCNPs nanoparticles. Then dissolve the αPECAM-1-HCNPs nanoparticles and antithrombin in equal proportions, and allow antithrombin to be adsorbed and loaded onto the αPECAM-1-HCNPs nanoparticles through electrostatic interaction, and purify to obtain αPECAM-1-AT@HCNPs; The preparation method of the HCNPs nanoparticles is as follows: Dissolve HES-CH in water and sonicate to obtain an aqueous solution of HES-CH. Dropwise add chloroform to this solution while sonicating, rotary evaporate to remove chloroform, centrifuge the obtained aqueous solution of HCNPs, discard the precipitate, and fully freeze-dry to obtain a powder of HCNPs nanoparticles.

2. The preparation method of the PECAM-1 targeted integrated diagnosis and treatment nanosensor according to claim 1, characterized in that, The radionuclide is 177 Lu; the reducing agent is carbodiimide hydrochloride and N-hydroxysuccinimide.

3. The preparation method of the PECAM-1 targeted integrated diagnosis and treatment nanosensor according to claim 1, characterized in that, In step S2: Add 177 PECAM-1_4G6 mAb labeled with Lu, and after stirring and reacting at room temperature, an aqueous solution of αPECAM-1-HCNPs nanoparticles is obtained.

4. The preparation method of the PECAM-1 targeted integrated diagnosis and treatment nanosensor according to claim 1, characterized in that, In step S2, after antithrombin is electrostatically adsorbed and loaded onto the αPECAM-1-HCNPs nanoparticles, ultrafiltration is performed to remove the unencapsulated and unloaded antithrombin to obtain purified αPECAM-1-AT@HCNPs.

5. The preparation method of the PECAM-1 targeted integrated diagnosis and treatment nanosensor according to claim 1, characterized in that, In step S2: The αPECAM-1-HCNPs nanoparticles and antithrombin are dissolved in ultrapure water in equal proportions and stirred to react, so that antithrombin is electrostatically loaded.

6. αPECAM-1-AT@HCNPs nanoparticles prepared by any of the methods of claims 1-5.

7. Use of the αPECAM-1-AT@HCNPs nanoparticles in claim 6 in the preparation of a product for detecting or inhibiting CRS occurring in CAR-T therapy.

8. Use of the αPECAM-1-AT@HCNPs nanoparticles in claim 6 in the preparation of a developer for CRS endothelial injury.

9. Use of the αPECAM-1-AT@HCNPs nanoparticles in claim 6 in the preparation of a drug for inhibiting CRS systemic inflammatory response diseases, wherein, the CRS systemic inflammatory response diseases include vascular endothelial injury and vascular barrier dysfunction.

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