CN111110874A - Radionuclide-labeled platelet membrane vesicle and preparation method and application thereof - Google Patents
Radionuclide-labeled platelet membrane vesicle and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a radionuclide-labeled platelet membrane vesicle and a preparation method and application thereof. Platelet membrane vesicle labeled nuclide125I、89Zr、45Ti、55Mn、59Fe、64Cu、94mTc、67Ga、71/72/74As、82Rb、89Y、124One or more of I. By utilizing the natural homing capability of the platelet membrane to atherosclerotic plaques, the platelet membrane can be used for in-situ detection of atherosclerosis after being labeled with nuclides, and has strong specificity, good biocompatibility and great application prospect.
Description
Technical Field
The invention relates to the field of cytobiology and in-vivo imaging tracing, in particular to a radionuclide-labeled platelet membrane vesicle and a preparation method and application thereof.
Background
Atherosclerosis (AS) is a chronic inflammatory disease and an important risk factor for causing severe cardiovascular and cerebrovascular events. The clinical outcome of atherosclerosis myocardial infarction and stroke are the two most common causes of death worldwide. However, atherosclerosis develops slowly and is usually only detected during the progressive phase due to the onset of symptoms and acute events. Therefore, early diagnosis and treatment of atherosclerotic plaques are of great importance.
The current commonly used imaging methods for diagnosing atherosclerotic plaques are: digital Subtraction Angiography (DSA), CT angiography (CTA), magnetic resonance imaging angiography (MRA), Carotid ultrasound, positron emission computed tomography (PET-CT), and Single photon emission computed tomography (SPET-CT) (Hassan M. Jonathan Gillard, Martin Graves, Thomas Hatsukaami, Chun Yuan (eds): cardio disease: thermal of imaging in diagnosis and management [ J ]. Pediatric ioradiology, 2007,37(4):406 and 408.). DSA is used for judging the degree of artery stenosis, which is the currently accepted gold standard for examining artery stenosis, but because it is an invasive examination, the radiation dose is large, and the conditions inside the vessel wall and the plaque cannot be estimated, the wide application of the DSA in clinic is limited. CTA can only display the amount of calcification and does not provide pathophysiological information in the atherosclerotic plaque, so that the CTA cannot be used for predicting vulnerable plaque and still lacks credible values for the prognosis, treatment effect evaluation and prognosis judgment of the plaque. Due to low resolution of MRA and complex sequence, small plaques are missed, and patients with contraindications (such as implanted devices and claustrophobia) cannot be diagnosed by MR imaging, so the clinical application of MR techniques is not high. The ultrasonic examination is currently the most widely used in clinical practice, and the subjective diagnosis of the operator has a great influence on the ultrasonic examination, so that the accuracy and repeatability of the ultrasonic examination are questioned, and the ultrasonic examination still has many aspects needing improvement as an objective judgment standard. SPET-CT and PET-CT are the most important methods of molecular imaging. The method can judge the metabolic conditions of different cells in the plaque on a molecular level, and can accurately position the plaque through the CT image, thereby providing great help in the aspects of treatment scheme selection, curative effect evaluation and prognosis judgment of the atheromatous plaque, and being the imaging method with the most advantages and potentials at present.
At present, the number of the current day,18f-deoxyglucose (C)18F-FDG) and18f-sodium fluoride (F-sodium fluoride)18F-NaF) is the most widely used PET-CT angiographic agent, especially18F-FDG is currently considered to be the gold standard for identifying atherosclerotic plaques by detecting vascular inflammation and macrophages. However,18the high myocardial uptake of F-FDG has severely affected its clinical utility. And the number of the first and second electrodes,18the specificity of F-FDG Imaging is limited, as is the spatial resolution of PET Imaging (. about.2 mm) (Minmin L, Hui T, Jun Z, et. bioengineered H-Ferritin Nanocages for Quantitative Imaging of Ulnerableplagues in Atherosclerosis [ J]ACS Nano,2018: acsnano.8b04158-. Therefore, the development of more specific radiotracers for imaging atherosclerosis is of great clinical value.
Platelets also play an important role in the development of atherosclerosis. Fluorescently labeled activated platelets were injected into ApoE knockout (ApoE-/-) mice, which were seen to home to atherosclerotic lesions. And studies have shown that the Platelet membrane-coated nanomaterial still has the property of Targeting atherosclerotic plaques (Wei X, YingM, Dehaini D, et al. nanoparticle function with plate membrane Enable Multi-factor Biological Targeting and protection of Atherosclerosis [ J]ACS Nano,2017 acsnano.7b07720.), thus using the characteristics of platelet membranes to naturally home atherosclerotic plaques, marked by the lodogen method125I, can be used as a contrast agent of SPET-CT to carry out specific targeted imaging on atherosclerotic plaques.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a radionuclide-labeled platelet membrane vesicle which can be used for specific targeted imaging of atherosclerotic plaques; the invention also provides a preparation method and application thereof.
The technical scheme is as follows: the invention relates to a platelet membrane vesicle labeled by radionuclide, and the platelet membrane vesicle labeled nuclide125I，89Zr、45Ti、55Mn、59Fe、64Cu、94mTc、67Ga、71/72/74As、82Rb、89Y、124One or more of I.
The average particle size of the platelet membrane vesicle is 100-110 nm.
The invention also provides a preparation method of the platelet membrane vesicle, which comprises the following steps: separating to obtain platelet membrane vesicles, and labeling the nuclide on the surfaces of the platelet membrane vesicles. The labeling method may be a conventional method, for example125I can be labeled by the lodogen method.
The platelet membrane vesicle can be mouse derived platelet membrane extracted from mouse platelet.
The invention also provides application of the platelet membrane vesicle in preparation of a developer for detecting atherosclerosis.
The invention also provides a developer containing the platelet membrane vesicle.
Wherein the imaging agent is useful for SPET/CT detection.
The imaging agent can be used for detecting atherosclerosis such as carotid atherosclerotic plaque.
Has the advantages that:
the platelet membrane has natural homing capacity on atherosclerosis, can target atherosclerotic plaques to realize in-situ detection after being labeled with nuclides, and has strong specificity.
The radionuclide-labeled platelet membrane vesicle can be used as or prepared into a contrast agent (developer) for SPET-CT detection of atherosclerosis.
The platelet membrane has good biocompatibility and great application prospect.
Drawings
FIG. 1 is a graph showing the particle size, potential and Transmission Electron Microscope (TEM) characterization of platelet membrane vesicles and erythrocyte membrane vesicles prepared in example 1 of the present invention; wherein A is a particle size diagram of the platelet membrane vesicle and the erythrocyte membrane vesicle, B is a potential diagram of the two, C is a TEM image of the platelet membrane vesicle, and D is a TEM image of the erythrocyte membrane vesicle;
FIG. 2 is a Coomassie brilliant blue staining pattern of platelet and platelet membrane vesicle proteins in example 1 of the present invention;
FIG. 3 is the platelet membrane vesicle labeling in example 1 of the present invention125I, high performance liquid chromatography characterization diagram;
FIG. 4 is a graph of gross, HE and oil red O staining of normal blood vessels and atherosclerotic blood vessels in example 2 of the present invention;
FIG. 5 shows the results of example 2 of the present invention125The SPET-CT imaging result of labeled platelet membrane vesicles and erythrocyte membrane vesicles, wherein A is a SPET-CT image of the erythrocyte membrane vesicles, B is a SPET-CT image of the platelet membrane vesicles, and C is a semi-quantitative image of signals of the A image and the B image at the position of a carotid artery; show that125The labeled platelet membrane vesicle can detect atherosclerosis in situ;
FIG. 6 is a sample taken in example 2 of the present invention125Three-dimensional cross-sectional view of SPET-CT imaging results at 1h for I-labeled platelet membrane vesicles.
Detailed Description
The invention will be further elucidated with reference to the following specific examples.
Example 1
This example provides a radionuclide125The preparation method of the I-labeled platelet membrane vesicle comprises the following steps:
(1) extraction of platelet membrane: taking 2ml of fresh mouse whole blood, adding 100ul of saturated EDTA-2Na solution for anticoagulation, centrifuging 300g for 5min to obtain supernatant to obtain platelet-rich plasma, and centrifuging 2000g of platelet-rich plasma for 5min to obtain platelet sediment. The platelet pellet was resuspended in PBS containing 10mM phosphatase inhibitor and 1mM protease inhibitor, frozen and thawed repeatedly in a freezer at-80 deg.C for 3 times, and finally centrifuged at 21000g for 30min to obtain a platelet membrane pellet. The obtained platelet membrane sediment is re-suspended by 200ul PBS containing phosphatase inhibitor and protease inhibitor, the solution is extruded by a 400nm polycarbonate membrane extruder, after 10 times of repetition, the solution is extruded by a 200nm polycarbonate membrane extruder, and platelet membrane vesicles are obtained after 10 times of repetition. The average particle size was measured by Malvern particle sizer to be 102nm and the average zeta potential was-28.5 mV, and the morphology was characterized by transmission electron microscopy, as shown in FIG. 1, wherein C is platelet membrane vesicles (PM) and D is erythrocyte membrane vesicles (RBCM).
Meanwhile, a hypotonic solution dissolution swelling breaking method is adopted to extract erythrocyte membranes as a control, and the specific operation steps are as follows: 2ml whole blood was centrifuged at 800g at 4 ℃ for 5 minutes, followed by careful removal of the serum and buffy coat, resuspension of the resulting red blood cell layer in 5ml pre-cooled 1 XPBS, centrifugation at 800g/5min to remove the pellet, and washing repeated 3 times. The washed erythrocytes are resuspended by precooled 0.25% PBS, incubated on ice for 20 minutes, centrifuged for 5min at 800g, hemoglobin is removed, repeated for a plurality of times until the supernatant after centrifugation is clear and transparent, finally, pink precipitates are collected and resuspended by 1ml PBS containing 10mM phosphatase inhibitor and 1mM protease inhibitor, and then the erythrocyte membrane vesicles are obtained by extruding the red erythrocytes by a polycarbofilm extruder with different pore diameters by the same method. The average particle diameter measured by Malvern particle sizer is 115nm, the average zeta potential is-30.5 mV, and the morphology is characterized by a transmission electron microscope, which is shown in figure 1 in detail, wherein C is a platelet membrane vesicle (PM) and D is an erythrocyte membrane vesicle (RBCM).
(2) Study of platelet membrane properties: in order to confirm that the platelet membrane vesicles still retain the membrane proteins of platelets, the whole proteins of platelet cells and platelet membrane vesicles are respectively extracted for SDS-PAGE electrophoresis, and Coomassie brilliant blue staining proves that the platelet membrane vesicles still retain the whole membrane proteins of platelet cells, which is shown in figure 2.
(3) Labeling on platelet membrane vesicles125I. Labeling by lodogen method on the premise that presence of intact membrane proteins in platelet membrane vesicles has been confirmed125I, high performance liquid chromatogram shows that iodine labeling is successful, see figure 3.
Experimental example 2 examination125Small animal SPET-CT imaging of I-labeled platelet membranes
(1) Experimental animals: female apolipoprotein E (ApoE)-/-The mice, 20, 8 weeks old, were purchased from the Qinglongshan animal breeding farm in Jiangning district, Nanjing, and randomly divided into 10 mice per group, each group being a model group and a control group. Model group mouse carotid atherosclerosis Model (Nam D, Ni C W, Rezvan A, et al.A Model of Distur) was constructed using high fat diet and carotid partial ligationbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by PartialLigation and a Simple Method of RNA Isolation from Carotid Endothelium[J]Journal of Visualized Experiments,2010(40). After the left carotid artery of the model group mice was partially ligated, high fat diet was started, and the normal group mice were fed normally. After 15 days, carotid arteries of 2 model mice and 2 Normal mice are randomly selected for gross tissue observation, HE staining and oil red O staining, and the results show that the left carotid intima layer of the model mice is broken, obvious lipid deposition occurs locally, and the model construction is proved to be successful, which is shown in figure 4, wherein a Normal group is a left side diagram and an untrament group is a right side diagram.
(2) And (5) collecting SPET-CT data of the small animal. Selecting 8 atherosclerosis model group mice as SPET-CT detection objects, and randomly selecting 3 tail veins for injection into 100 microorgans125I-labeled platelet membrane was used as experimental group, and 3 tail vein injections of 100 microorgans were randomly selected125I-labeled erythrocyte membranes served as control group. Whole body scanning images of mouse SPET-CT 5min, 30min, 1h, 1.5h and 2h after injection are respectively collected. The result shows that the time is 5min125The platelet membrane marked with I begins to show signals in the atherosclerotic region, the signals gradually increase at 30min, the signals reach the maximum at 1h, the signals disappear at 2h, and125the I-labeled erythrocyte membrane has no relevant signal. Confirmation of125The platelet membrane marked by the I can be specifically targeted to an atherosclerosis area, can be used for in-situ detection of atherosclerosis, and has strong specificity and good biocompatibility. The results are shown in FIGS. 5 and 6. FIG. 5 is a SPET-CT plot of erythrocyte membrane vesicles at A, a SPET-CT plot of platelet membrane vesicles at B, and a semi-quantitative plot of the signal at carotid artery location for A and B plots; FIG. 6 is a graph of medium acquisition125Three-dimensional cross-sectional view of SPET-CT imaging results at 1h for I-labeled platelet membrane vesicles.
Claims (8)
1. A platelet membrane vesicle labeled with a radionuclide, characterized in that the platelet membrane vesicle is labeled with a nuclide125I、89Zr、45Ti、55Mn、59Fe、64Cu、94mTc、67Ga、71/72/74As、82Rb、89Y、124One or more of I.
2. The platelet membrane vesicle according to claim 1, wherein the mean particle size of the platelet membrane vesicle is 100-110 nm.
3. The platelet membrane vesicle according to claim 1, wherein the platelet membrane vesicle is of murine origin.
4. The method for producing platelet membrane vesicles according to any one of claims 1 to 3, comprising: separating to obtain platelet membrane vesicles, and labeling the nuclide on the surfaces of the platelet membrane vesicles.
5. Use of platelet membrane vesicles according to any one of claims 1 to 3 in the preparation of a contrast agent for the detection of atherosclerosis.
6. An imaging agent comprising the platelet membrane vesicle according to claim 1 or 2.
7. The imaging agent of claim 6, wherein the imaging agent is used for SPET/CT detection.
8. The imaging agent according to claim 6, wherein the imaging agent is used for detecting atherosclerosis.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108042510A (en) * | 2018-02-11 | 2018-05-18 | 复旦大学附属中山医院 | A kind of coated rapamycin bionic nano particle of platelet membrane for targeting atherosclerotic plaque and application thereof |
| CN110339180A (en) * | 2019-08-12 | 2019-10-18 | 苏州大学 | A kind of anti-inflammatory targeted delivery systems and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108042510A (en) * | 2018-02-11 | 2018-05-18 | 复旦大学附属中山医院 | A kind of coated rapamycin bionic nano particle of platelet membrane for targeting atherosclerotic plaque and application thereof |
| CN110339180A (en) * | 2019-08-12 | 2019-10-18 | 苏州大学 | A kind of anti-inflammatory targeted delivery systems and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| HARALD F. LANGER ET AL.: "Radionuclide Imaging A Molecular Key to the Atherosclerotic Plaque", 《JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY》 * |
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