CN113229213B - A method for pulmonary embolism modeling and non-invasive quantitative detection by labeling thrombus with near-infrared fluorescent probes - Google Patents

Abstract

The invention relates to a near-infrared fluorescent probe molecule that can be combined with thromboembolism and its application in modeling of pulmonary embolism and non-invasive quantitative detection. In this method, traceable thromboembolism is formed in vitro from rat plasma, thrombin, calcium chloride and near-infrared fluorescent probe molecules, which are chopped and ground to a micron scale, and then injected into mice through the tail vein. . The thromboembolic particles can accumulate in the pulmonary blood vessels of mice, and then block the blood vessels to form embolism, realizing the establishment of a mouse pulmonary embolism model, supplemented by a small animal in vivo fluorescence imaging system to achieve non-invasive quantitative detection, and can be applied to thrombolytic drug dissolution Evaluating the effect of embolization, etc.

Description

A method for pulmonary embolism modeling and non-invasive quantitative detection by labeling thrombus with near-infrared fluorescent probes

technical field

The invention relates to the field of biomedicine, in particular to a mouse pulmonary embolism model constructed by using near-infrared fluorescent probe-labeled thromboembolic particles and its application.

Background technique

Pulmonary embolism (PE) is a cardiovascular emergency caused by obstruction of the pulmonary artery or its branches by exogenous or endogenous emboli, which seriously threatens human life and health. Right ventricular failure, circulatory shock, and cardiac arrest secondary to acute PE are responsible for its high mortality. In recent years, the number of PE patients in my country has been increasing year by year. With early diagnosis and timely treatment, the fatality rate of patients with acute PE can be reduced from 30% to 8%. Treatment options for acute PE include anticoagulation, thrombolysis, interventional therapy, and surgery. Thrombolytic therapy is a drug that directly or indirectly converts the inactive plasma protein plasminogen into activated plasmin, which acts on the fibrin clot, causing it to dissolve, while removing and inactivating coagulation factor II. , Ⅴ, Ⅷ, interfere with coagulation and play anticoagulation. In recent years, many researchers have devoted themselves to the development of new thrombolytic drugs, and a number of convincing research results have provided evidence-based medical evidence for thrombolytic therapy in acute PE. Therefore, it is urgent to develop a simple, effective, and reproducible standardized model of pulmonary embolism, which can be used to further study the molecular mechanism of pulmonary embolism and evaluate the thrombolytic efficiency of different thrombolytic regimens, so as to reduce the bleeding complications of thrombolytic drugs. , benefiting patients.

At present, several mouse PE models have been constructed to simulate human pulmonary obstructive thromboembolism, such as coagulation factor-induced mouse PE model, photochemical reaction-induced mouse PE model, and exogenous thrombus injection-induced small PE model. Murine PE model. The mouse PE model induced by coagulation factors has the advantages of simple operation and low price, and has been widely used to detect the antithrombotic effect of compounds in vivo. However, coagulation factors frequently lead to fatal thromboembolism, and the pathophysiology of pulmonary embolism induced by them is different from PE secondary to venous thrombosis in humans. The mouse PE model induced by photochemical reaction uses Rose Bengal B to generate singlet oxygen free radicals under green light irradiation, which damages the vascular endothelium, causes platelet adhesion, stimulates the coagulation process, and leads to the formation of intravascular thrombosis in the irradiation area. The PE model established by this method is closer to PE that occurs under human pathophysiological conditions, but the thrombus formed is more common in the pulmonary microvessels, but rare in the large pulmonary vessels. The mouse PE model induced by exogenous thrombus injection is to inject blood clots prepared in vitro into mice through the tail vein or jugular vein. It dissolves spontaneously and slowly under physiological conditions, and is not lethal.

The rapid development of in vivo imaging technology in living animals has greatly promoted the diagnosis and treatment of diseases and the evaluation of drug activity at the in vivo level, and has also been used for the evaluation of thrombolytic drugs. In vivo imaging techniques of living animals are mainly divided into optical imaging (optical imaging), radio-nuclear imaging (radio-nuclear imaging), magnetic resonance imaging (magnetic resonance imaging, MRI), ultrasound imaging (ultrasound imaging), computed tomography (computed tomography, CT) . With the improvement of spatiotemporal resolution of multi-slice spiral CT, computed tomographic pulmonary angiography (CTPA) has replaced angiography and has become the gold standard for clinical diagnosis of PE. Optical imaging has the unique advantages of simple operation, intuitive results, fast measurement, high sensitivity, low cost, repeatability, and long-term monitoring. It is more advantageous in drug research and screening.

SUMMARY OF THE INVENTION

The purpose of the present invention is to establish a mouse PE model induced by exogenous fluorescently labeled thrombus, which can use optical imaging to trace the fluorescently labeled thrombus for evaluation of the thrombolytic effect of drugs.

Pulmonary thromboembolism (PTE) is the most common type of acute PE. It is caused by thrombus from the venous system or right heart blocking the pulmonary artery or its branches. The main pathophysiological features and clinical manifestations are pulmonary circulation and respiratory dysfunction. It accounts for the vast majority of acute PE and is the most severe clinical manifestation of venous thromboembolism (VTE), and in many cases it is secondary to deep venous thrombosis (DVT).

In order to better simulate the natural occurrence and development process of PTE, the establishment of animal models is mainly through in vitro injection of embolic particles. Specifically, blood clot embolic particles prepared in vitro are injected into veins, and then incarcerated in the pulmonary artery through blood circulation. , forming a PE model. When the constructed PE model is used to evaluate the thrombolytic effect of thrombolytic drugs, in order to reduce the number of animals used and realize the real-time monitoring of the thrombolytic process, the embolic particles prepared in vitro need to have traceable properties.

The present invention is achieved by the following technical solutions: a near-infrared fluorescent probe molecule that can bind to thromboembolism is pentalysine β-carbonyl zinc phthalocyanine (ZnPc-(Lys) ₅ ), the structure of which is shown in the figure As shown in Figure 1, the pentalysine modification increases the hydrophilicity of the compound molecule and makes it positively charged, which can be combined with the negatively charged components in the thrombus to target the thrombus.

Further, the present invention provides a traceable thromboembolic particle, and the particle size of the thromboembolic particle is about 1-5 microns. The preparation method of the thromboembolic particles is as follows: using rat plasma, adding fluorescent probe pentalysine beta-carbonyl phthalocyanine zinc, and using thrombin and calcium chloride as coagulants, the formed fluorescent label is The thrombus was shredded and ground moderately to particle size with a mortar and suspended in normal saline. The main components of the traceable thromboembolism particles formed by grinding are highly cross-linked fibrin and platelets. The emboli have a dense structure and are not easily destroyed by the animal's own fibrinolytic system in vivo, resulting in self-lysis of the emboli.

Further, the present invention provides a method for constructing a novel mouse PE model, which is characterized in that the fluorescently traceable thromboembolic particles formed in vitro are injected into the mouse body through the tail vein, and accumulated in the pulmonary artery through blood circulation, Causes pulmonary artery blockage. This process is close to the natural occurrence and development of PTE under pathophysiological conditions.

Further, the present invention provides a strategy for evaluating the thrombolytic effect of a thrombolytic drug, which is characterized in that the signal of the near-infrared fluorescent probe in the mouse lung represents the accumulation and blockage level of thromboembolic particles in the lung blood vessels of the mouse. Therefore, the thrombolytic process can be characterized and the effect of thrombolytic drugs can be evaluated by the real-time quantitative fluorescent probe concentration in the lungs of mice.

Compared with the existing small animal PE models that can be used to evaluate and screen thrombolytic drugs, the innovation and special features of the present invention are:

(1) The particle size of the thromboembolic particles prepared in vitro by the present invention is about 1-5 microns, the surface is negatively charged, and can be targeted and labeled by positively charged ZnPc-(Lys) ₅ . Under the excitation of 680 nm light source, it can produce Strong fluorescence signal. The mouse modeling method is simple and close to the occurrence and development of PTE in the pathological state in vivo, and the subsequent non-invasive and real-time quantitative monitoring can be performed by FMT.

(2) After injection through the tail vein, the thromboembolic particles can be evenly distributed in the pulmonary artery of mice, and are not easily dissolved by the mouse's own fibrinolytic system, and can accumulate stably in the pulmonary artery for more than 6 hours, which provides a basis for the subsequent evaluation of the effect of thrombolytic drugs. a sufficiently long time window.

(3) The mouse PE model constructed by the present invention can be used for in vivo evaluation and screening of novel thrombolytic agents, and has the advantages of high efficiency, reliability, and low cost.

In summary, the present invention has developed a method for constructing a mouse PTE model by injecting thromboembolic particles labeled with a near-infrared fluorescent probe through the tail vein, which greatly simplifies the experimental operation process of the mouse PTE model. Data variability was low and there were no deaths. In addition, the application of in vivo 3D real-time quantitative imaging has greatly reduced the number of small animals required to evaluate thrombolytic effects, which could facilitate current research on PE interventions.

Description of drawings

Fig. 1 is the chemical structure of ZnPc-(Lys) ₅ ;

Figure 2 shows the preparation and characterization of ZnPc-(Lys) ₅ -labeled thromboembolic particles; wherein:

(A) Picture of ZnPc-(Lys) ₅ -labeled thromboembolism formed in vitro;

(B) Size distribution of ZnPc-(Lys) ₅ -labeled thromboembolic particles;

(C) Surface zeta potential of ZnPc-(Lys) ₅ -labeled thromboembolic particles;

(D) UV-Vis absorption spectrum of ZnPc-(Lys) ₅ -labeled thromboembolic particles;

(E) Fluorescence spectra of ZnPc-(Lys) ₅ -labeled thromboembolic particles;

Figure 3 shows thrombolysis of thromboembolism and embolic microparticles in vitro; wherein:

(A) Thrombolysis results of unground thrombus emboli treated with different concentrations of r-tPA;

(B) Changes in the average particle size of the main particles after the ground thromboembolic particles were treated with 200 nM r-tPA;

Figure 4 shows the construction and FMT characterization of the mouse PE model; wherein:

(A) 3D imaging results of accumulation and distribution of free fluorescent probe group ZnPc-(Lys) ₅ in mouse lung;

(B) 3D imaging results of accumulation and distribution of ZnPc-(Lys) ₅ in mouse lungs in PE model group;

(C) Quantification of the concentration of ZnPc-(Lys) ₅ in mouse lungs in the free fluorescent probe group and PE model group;

Fig. 5 is r-tPA thrombolytic effect evaluation; Wherein:

(A) 3D imaging results of the accumulation and distribution of ZnPc-(Lys) ₅ in the lungs of mice in the saline-treated group and the r-tPA-treated group;

(B) Quantification of the concentration of ZnPc-(Lys) ₅ in the lungs of mice in the saline-treated and r-tPA-treated groups (**** represents P <0.0001);

Figure 6 shows the 2D imaging results of isolated lung tissue of mice in different experimental groups;

Figure 7 shows the results of H&E staining of isolated lung tissue sections of mice in different experimental groups (magnification 10×, scale bar: 300 μm).

Detailed ways

Example 1: Preparation and Characterization of ZnPc-(Lys) ₅ Labeled Thromboembolic Microparticles

(1) Preparation of ZnPc-(Lys) ₅ -labeled thromboembolic particles: SD rat orbital blood was taken, anticoagulated with 3.2% sodium citrate (the volume ratio of anticoagulant to whole blood was 1:9), Whole blood was centrifuged at 1200 g for 10 min to separate plasma. Take 500 μL of plasma and add 2 μL ZnPc-(Lys) ₅ (4.5 mM), 11.5 μL thrombin (10 U/mL) and 46.5 μL calcium chloride (240 mM), mix well and incubate in a 37 °C oven for 2 h . The formed ZnPc-(Lys) ₅ -labeled thrombosis was placed in a mortar, cut into small pieces and resuspended in 1 mL of Tyrodes-Hepes buffer (THB), and ground for 10 min to form thromboembolic particles.

(2) Characterization of ZnPc-(Lys) ₅ -labeled thromboembolic particles: 100 μL of the prepared fluorescently-labeled thromboembolic particles were randomly sampled and placed in a 96-well plate, and placed in a Spectra Max i3x microplate reader to scan them. UV-Vis absorption spectrum (400-800 nm, 2 nm/read) and fluorescence spectrum (excitation wavelength: 610 nm, scanning emission wavelength 600-800 nm, 2 nm/read). In addition, 1 mL of fluorescently labeled thromboembolic particles were randomly sampled, centrifuged at 13,000 rpm for 10 min, and then resuspended in deionized water. Dynamic light scattering (Zetasizer Nano-ZS, Malvern, PA, USA) was used to determine their particle size distribution and surface zeta Potential, fixed scattering angle of 90 degrees, equilibration time of 90 s, and repeated the measurement 3 times.

The results are shown in Figure 2. The particle size distribution of ZnPc-(Lys) ₅ -labeled thromboembolic particles showed three peaks, of which 80% of the particles had an average particle size of 1.2 μm, 16% of the particles had an average particle size of 5 μm, and 4% of the particles had an average particle size of 1.2 μm. The average particle size is 100 nm. The surface of unlabeled thromboembolic particles was strongly negatively charged, which was partially neutralized after being labeled with positively charged ZnPc-(Lys) ₅ . ZnPc-(Lys) ₅ -labeled thromboembolic particles exhibited characteristic absorption at 680 nm and a strong fluorescence signal in the near-infrared region. However, ZnPc-(Lys) ₅ dissolved in THB buffer has a maximum absorption at 630 nm due to aggregation and no fluorescence in the near-infrared region.

Example 2: In vitro thrombolysis of unground and ground thromboembolic microparticles

(1) In vitro thrombolysis of unground thromboembolism: blood was collected from the orbit of SD rats, anticoagulated with 3.2% sodium citrate (the volume ratio of anticoagulant to whole blood was 1:9), and the whole blood was treated with 1200 g Plasma was separated by centrifugation for 10 min. Take 30 μL of plasma and add it to a 96-well plate, followed by adding 123.3 μL Tris buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and 6.7 μL calcium chloride (240 mM), mix well and store in an oven at 37 °C Incubate for 2 h. After thromboembolism was formed, different concentrations of r-tPA were added for thrombolysis, and the absorption at 405 nm was recorded in a Spectra Max i3x microplate reader.

(2) In vitro thrombolysis of ground thrombosis: The unground thrombosis formed above was taken out with micro tweezers, placed in a mortar, cut into small pieces, and resuspended by adding 0.2 mL of Tyrodes-Hepes buffer (THB). Suspended and ground for 10 min to form thromboembolic particles. The particle size distribution was determined by dynamic light scattering (Zetasizer Nano-ZS, Malvern, PA, USA). The final concentration of 200 nM r-tPA was added for thrombolysis, and the particle size changes of thromboembolic particles at different time points were detected.

The results are shown in Figure 3, and the absorption at 405 nm of the unground thromboembolus remained unchanged in the absence of r-tPA treatment. The thrombolytic effect of r-tPA is dose-dependent, 10 nM r-tPA and thrombus co-incubation for 2 h can only partially dissolve thrombosis, and 50 nM r-tPA and 100 min co-incubation of thromboembolism can make the thrombosis Complete dissolution, 100 nM r-tPA incubated with thrombus for 60 min can make it completely dissolved, 200 nM r-tPA incubated with 50 min of thromboembolism can make it completely dissolved. For the ground thromboembolic particles, 80% of which have an average particle size of 1.2 μm, co-incubating with r-tPA with a final concentration of 200 nM can gradually reduce the average particle size of the thromboembolic particles with time, and the total After incubation for 6 h, the average particle size decreased to about 700 nm. The results showed that the particle structure of thromboembolism formed by grinding was dense and difficult to be completely dissolved.

Example 3: Construction of mouse PE model and in vivo optical imaging

The prepared ZnPc-(Lys) ₅ -labeled thromboembolic particles were injected into ICR mice through the tail vein (200 μL/20 g). The fluorescence signal of ZnPc-(Lys) ₅ in mouse lungs was monitored in real time in an in vivo animal imager (FMT2500 ^TM LX instrument, PerkinElmer). The instrument uses a 680 nm laser diode to excite ZnPc-(Lys) ₅ molecules, selects mouse lungs as Regions of interest (ROIs), and scans 50-60 source locations (3 mm between adjacent scan points). At the same time, the same concentration of free ZnPc-(Lys) ₅ solution was injected into mice as a control (free fluorescent probe group). To quantify the concentration of ZnPc-(Lys) ₅ , the FMT instrument was calibrated with 1 μM ZnPc-(Lys) ₅ (dissolved in DMSO) as a standard. The recorded images were 3D reconstructed by TrueQuant v3.0 software, and quantitative results were obtained.

The results are shown in Figure 4. Free ZnPc-(Lys) ₅ and ZnPc-(Lys) ₅ -labeled thromboembolic particles were injected into mice through tail vein respectively (free fluorescent probe group and PE model group, respectively) , FMT imaging results showed that ZnPc-(Lys) ₅ -labeled thromboembolic particles rapidly accumulated in the lungs, reaching the highest concentration in the lungs 1 h after injection, and then gradually decreasing their concentrations in the lungs; Only a small amount of free ZnPc-(Lys) ₅ was taken up into the lung tissue, and the concentration of free ZnPc-(Lys) ₅ in the lungs at each time point was significantly lower than the concentration of ZnPc-(Lys) ₅ in the PE model group ( P < 0.001), indicating that the fluorescence signal of the lung displayed by FMT imaging in the PE model group was mainly caused by the accumulation of thromboembolic particles in the pulmonary artery.

Example 4: The mouse PE model is used for the evaluation of the thrombolytic effect of r-tPA

The PE model mice constructed by intravenous injection of ZnPc-(Lys) ₅ -labeled thromboembolic particles were randomly divided into two groups (12 mice in each group). tPA (10 mg/kg), and the fluorescence signal of ZnPc-(Lys) ₅ in mouse lungs was monitored at 30 min, 1 h, 3 h, and 6 h, respectively, and the concentration of ZnPc-(Lys) ₅ was used to characterize thromboembolic particles The thrombolytic effect of r-tPA was evaluated in terms of accumulation and blockage in the lungs.

The results are shown in Figure 5. The FMT imaging results showed that compared with the normal saline control group, the fluorescence intensity accumulated in the lungs of the mice treated with r-tPA decreased significantly at 1 h, 3 h, and 6 h ( P < 0.001), indicating that r-tPA produced a thrombolytic effect.

Example 5: 2D imaging of ex vivo tissue and staining of tissue sections to verify the applicability of mouse PE model for screening of thrombolytic agents

At each of the above time points (30 min, 1 h, 3 h, and 6 h), three mice in each group (free fluorescent probe group, saline-treated group, and r-tPA-treated group) were sacrificed by cervical dislocation and dissected. The lung tissue samples were then taken out, rinsed with normal saline, and then drained, and placed in the FMT instrument for 2D imaging. After that, the lung tissue was fixed in 4% paraformaldehyde, dehydrated successively, embedded in paraffin, and sliced. The slices were deparaffinized, rehydrated, and stained with hematoxylin and eosin.

The results are shown in Figure 6. The 2D imaging results of the lung tissue of the mice in each group showed that the fluorescence in the lungs of the mice in the free probe group was very weak at each time point, and the fluorescence in the lungs of the mice in the PE model group was stronger, especially in the modeling After 1 h and 3 h, the fluorescence intensity was significantly stronger than that in the r-tPA treatment group, while the lung fluorescence of the mice in the r-tPA treatment group was strong at 1 h, and then the fluorescence intensity decreased rapidly.

The staining results of lung tissue sections are shown in Figure 7. Obvious thromboembolism was seen in the pulmonary blood vessels of the mice in the saline-treated group and the r-tPA-treated group at 1 h, while the pulmonary blood vessels of the r-tPA-treated mice at 3 h The internal emboli were dissolved, and at 6 h, the pulmonary thrombus emboli in the pulmonary blood vessels of the mice in the r-tPA treatment group were significantly reduced compared with the mice in the normal saline control group. It is further verified that the PE model constructed by the present invention and the evaluation platform based on optical imaging are suitable for the development and screening of new thrombolytic agents.

The specific embodiments described above are further detailed descriptions of the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be Included within the scope of protection of the present invention.

Claims (3)

1. An exogenously traceable thromboembolic microparticle, characterized by: labeling thrombus embolus by utilizing near-infrared fluorescent probe molecules, wherein the particle size of thrombus embolus particles is 1-5 microns; the near-infrared fluorescent probe molecule is penta-polylysine beta-carbonyl zinc phthalocyanine, and the structural formula is as follows:

；

the preparation method of the thrombus embolic particles comprises the following steps: a fluorescence probe, namely pentalysine beta-carbonyl zinc phthalocyanine is added into rat plasma, thrombin and calcium chloride are used as coagulants, formed fluorescence-labeled thrombus is cut into pieces, ground by a mortar and suspended in physiological saline.

2. A noninvasive quantitative detection method of a mouse pulmonary embolism model is characterized by comprising the following steps: carrying out living body imaging of the small animals by fluorescent molecule tomography, exciting near-infrared fluorescent probe molecules by using a laser diode with the wavelength of 680 nm, and obtaining a quantitative result of the probe molecules by three-dimensional reconstruction; the mouse pulmonary embolism model is characterized in that exogenous traceable thromboembolism particles are injected into a mouse body through tail veins to accumulate and block blood vessels in the mouse lung to cause pulmonary embolism.

3. The application of the method for non-invasive quantitative detection of the mouse pulmonary embolism model according to claim 2 in evaluating the thrombolytic effect of a thrombolytic drug, wherein the method comprises the following steps: after the thrombolytic drug is given, the accumulation condition of the fluorescent probe in the lung of the mouse is monitored in real time through a small animal living body imaging system and quantified, the concentration of the fluorescent probe in the lung reflects the dissolution degree of the thrombolytic particles, and the lower the concentration of the fluorescent probe is, the better the thrombolytic effect of the thrombolytic drug is.

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