KR20120132362A - Method for monitoring CT-based customized to individual thrombus image using gold nanoparticle - Google Patents

Abstract

PURPOSE: A CT(Computed Tomography)-based monitoring method customized to an individual thrombus using a gold nanoparticle for optimizing determination of thrombolytic drug dosage is provided to obtain a CT image informing size and location of a thrombus, thereby noninvasively monitoring a thrombus not sacrificing experimented animals. CONSTITUTION: A gold nanoparticle coated with glycol chitosan is injected into an animal. The gold nanoparticle coated with glycol chitosan is made from reaction between glycol chitosan solution and gold chloride solution. A CT image is obtained by a micro CT. The CT image is reconstructed into a 3D(Three Dimensional) image using software.

Description

Method for monitoring CT-based customized to individual thrombus image using gold nanoparticles

The present invention relates to a CT-based patient-specific thrombosis monitoring method using gold nanoparticles.

Arteries, veins, cardiac and thrombosis in the cardiovascular system, in particular, cause many diseases such as stroke, heart attack, numbness, deep vein thrombosis and pulmonary embolism. Among them, stroke-induced acute thrombosis and acute carotid artery thrombosis, which causes acute cerebral infarction, have become a major social problem, such as the recent proliferation of the disease with the development of modern society.

In addition, in economic terms, the total cost of such strokes is more than $ 10 billion annually worldwide, for example in hospitals and nursing care, hospitals, doctors and health care professionals, medicine, home health costs and other medical consumer goods. There are direct costs involved, or indirect costs such as lost productivity due to illness, and lost productivity due to mortality.

Efforts to prevent and treat stroke, a major social issue on health, have evolved over the last 60 years through many studies, including angiography, artificial heart valves, and computed tomography for differential diagnosis. CT), Transcranial Doppeler (TCD), and the like have been studied.

In addition, conventional studies have recently emerged in which techniques involving the administration of thrombolytic agents such as tissue tissue plasminogen activators (tPA) have been shown to reduce neurobiological damage.

In Korea, approximately 100,000 acute strokes (about 80% of which are cerebral infarction) occur each year, and about 3 to 4% of patients are reported to receive tPA treatment. Only Korea's economic benefits from its use are estimated at about 20 billion won every year.

According to the NINDS tPA test, in conventional thrombolytic treatment, the probability of living independently without the help of one person is one in seven treatments, but also within three to six hours of symptoms. Only thrombosis has been found to have the problem that can be treated.

These problems seem to be due to the fact that the effectiveness of thrombolytic treatment is mild to moderate effect, and that 5 to 10% of the thrombolytic treatments do not have low side effects such as cerebral hemorrhage or brain edema. Therefore, if the side effects can be lowered while increasing the effect of thrombolytic treatment even a little, and if the extent of the treatment target can be expanded, the humanitarian benefits of the stroke condition to patients and their families, additional countries, Economic benefits are thought to be very large.

On the other hand, in the monitoring of thrombus, the prognosis of the patient is poor as the delay of the treatment for the patient is delayed. Therefore, CT, which can acquire a brain image faster than MRI, in particular, does not use a contrast agent. Non-contrast CT) is the most widely used imaging tool for monitoring thrombus.

When using non-contrast CT, the location and size of the thrombus are often not known. Therefore, it is not a tailored therapy in the method of monitoring the thrombus such as the dose of the thrombolytic drug or the decision of the drug injection or procedure. Serious problems can arise that can only be relied on by a pre-determined fixed protocol.

In addition, MRI can be used to monitor the effect of thrombolytic activity without sacrificing experimental animals, but it is difficult to quantify and much more expensive than CT, which makes it difficult to conduct large-scale and quantitative experiments. There is an urgent need for a method to solve this problem.

Accordingly, the present inventors, while continuing to make efforts to solve the conventional problems as described above, when rapidly monitoring the position and size of the thrombus, through this it is possible to optimize the dose determination of the thrombolytic drug through the patient-specific thrombus By discovering that treatment is possible, the present invention has been completed.

The present invention is to provide a CT-based patient-specific thrombosis monitoring method using gold nanoparticles.

The present invention provides a CT-based thrombus imaging monitoring method using gold nanoparticles coated with glycol chitosan as a CT contrast agent.

The thrombus monitoring method according to the present invention enables the rapid and repetitive acquisition of CT image information on the size and location of thrombus in cardiovascular thrombosis, and by using this, new drug and new drug candidates for cardiovascular system diseases including thrombolytic agents. The biological effect of the non-invasive monitoring is possible without sacrificing the experimental animal.

1 is a diagram showing the appearance of thrombosis after the carotid artery exposure (left) and thus thrombosis in the mouse model (right).
2 is a diagram showing the size and location of the thrombus in the normal artery (A) and the blood vessel (B) clogged tightly when angiography is performed after thrombosis induction.
FIG. 3 is a diagram tracking the location and size of thrombi from 5 minutes after injection of gold nanoparticles to 48 hours.
FIG. 4 is a diagram illustrating a living blood clot image proportional to histological thrombus size. FIG.
5 is a diagram showing the results of quantitation of thrombus seen in CT images according to histological thrombus size (n = 35 per group).
6 is a diagram showing the results of quantitation of thrombus size seen in time-phase CT images according to histological thrombus size (n = 5 per group).
7 is a view showing that once-injected gold nanoparticles can repeatedly diagnose recurrent thrombosis.
8 is a diagram quantifying the longest length, thrombus volume, thrombus CT density and amount of gold nanoparticles in thrombi of CT images obtained 5 minutes after injection of gold nanoparticles 30 minutes after thrombosis induction (n = per group) 64).
9 is a diagram showing that after the thrombolytic treatment, a portion of the thrombus moves to the distal blood vessel and changes its position while the thrombus melts.
10 is a diagram showing that the thrombus volume is significantly reduced on the CT according to the thrombolysis.
FIG. 11 shows quantitative results showing significant decrease in thrombus volume on CT as thrombi lysis (n = 5 per group).
FIG. 12 shows quantitative results showing that volume reduction of thrombi on CT is different according to the thrombolytic treatment protocol.

The present invention provides a CT-based thrombus imaging monitoring method using gold nanoparticles coated with glycol chitosan as a CT contrast agent.

Hereinafter, the present invention will be described in detail.

The thrombus imaging monitoring method of the present invention is characterized in that after the injection of gold nanoparticles coated with glycol chitosan to animals other than humans with thrombosis, CT images are obtained by using micro CT.

As used herein, the term 'thrombosis' is a disease caused by 'thrombosis', which is a lump of blood in a blood vessel, and is also referred to as thromboembolism, and particularly refers to a disease in which a blood vessel is blocked by a thrombus. Our body is balanced with a number of thrombogenic factors and regulators, so that normal blood clots don't make excessive blood clots. However, if the balance of factors involved in thrombus formation inhibition is broken, a thrombus may be formed. The causes of the thrombosis are three cases of slow blood flow, excessive coagulation, and blood vessel damage. These three causes, either alone or in combination, are a direct cause of thrombosis. A wide variety of symptoms can occur depending on the location of the organ where the thrombosis occurs and the types of blood vessels. Arterial thrombosis is mainly caused by ischemic symptoms that may occur when blood is not supplied properly and peripheral blood flow is insufficient. The main cause is congestion or congestion, which can be caused by blood reaching the end but not returning to the heart. The presence of blood clots is diagnosed by diagnosing the presence of blood clots in the body parts suspected of thrombosis. Ultrasound, CT, MRI, angiography, and radioisotope scans can be used to confirm the presence of blood clots. In the present invention, the thrombosis image based on CT is monitored.

The thrombosis includes, but is not limited to, thrombosis, cardiovascular thrombosis, or peripheral vascular thrombosis.

The 'cerebral thrombosis' refers to a disease in which a part of the cerebral blood vessels is caused by an arterial stenosis or a blood clot caused by a blood clot, and most of the cerebral thrombosis is caused by a pleural lesion of the brain. do. The mechanism of thrombus formation due to atherosclerosis is not known yet, but in addition to ulcers, edema, and bleeding in the hardened area, blood flow is delayed, and thus systemic effects such as lowering blood pressure and hypercoagulation are added. As for cerebral vascular changes, cerebral vascular changes occur most in the branches of the middle cerebral artery, followed by the rankings of the anterior and posterior cerebral arteries. It is likely to occur in people over 60 years of age or high blood pressure. However, the relationship between cerebral hemorrhage and hypertension is not close. Vascular disorders often occur in parts of the body other than the brain, for example, due to myocardial disorders and inability to touch the lower extremities.

The cardiovascular or cardiovascular disease refers to diseases occurring in the heart and major arteries. Heart disease is a congenital heart disease from birth and acquired heart disease that occurs in life.Heart disease is a disease that occurs in each region, as the structure of the heart can be divided into heart muscle, cardiovascular, valve, and conduction system that is responsible for the electrical signals of the heart. Can be classified. Major vascular diseases are those in which major arteries such as the aorta, ischemia, carotid arteries, cerebrovascular, renal arteries, and lower extremity arteries (such as the articular iliac artery and thigh) are blocked, stretched, or bleeding to be.

Gold nanoparticles coated with glycol chitosan may be prepared by reacting an aqueous solution of glycol chitosan with an aqueous solution of gold salt (HAuCl ₄ ).

The shape and size of the gold nanoparticles can be controlled by adjusting the concentration of the gold salt solution and the amount of glycol chitosan, the average particle diameter of the gold nanoparticles used in the present invention is preferably about 18 ~ 22 nm.

Gold nanoparticles coated with glycol chitosan used in the present invention have excellent performance that stability is maintained for more than 45 days.

Gold nanoparticles coated with glycol chitosan of the present invention (0.5 ~ 1.5 mg / ml) may be injected in an amount of 90 ~ 110 μl.

The term 'CT (computed tomography)' used in the present invention is to collect the X-rays transmitted by irradiating the target site of the human body from various directions with a detector, and to detect the difference in absorption of X-rays for the site. A photography technique in which computers reconstruct using mathematical techniques. CT has excellent resolution and contrast in distinguishing blood, cerebrospinal fluid, white matter, gray matter, and tumors compared to conventional X-ray images, and can represent minute differences in absorption. .

The present invention solves the problem of the conventional contrast agent was not able to accurately determine the size or location of the blood clot in the blood vessel is clogged blood clots tightly, imaging a stroke-induced acute thrombosis in 5 to 15 minutes, the size of the patient's blood clots And then, the patient can provide a customized monitoring method according to the location and size of the thrombus.

The blood clots monitored as described above can be efficiently treated by adjusting the type and amount of the thrombolytic agent according to the position and size.

Therefore, the thrombus monitoring method according to the present invention enables the rapid and repetitive acquisition of CT image information on the size and location of thrombus in cardiovascular thrombosis. The in vivo effect of the material allows for non-invasive monitoring without sacrificing experimental animals.

Animals other than humans in the present invention include, but are not limited to, dogs, mice, cats, monkeys, sheep, goats, cattle, horses, and pigs.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are only illustrative of the present invention, and the scope of the present invention is not limited thereby.

Preparation Example 1 . Glycol  Gold nanoparticles coated with chitosan ( GC - AuNP Manufacturing

After dissolving 300 mg of glycol chitosan (molecular weight 250,000) in 300 ml of water, the solution was filtered through a filter (pore size: 0.45 μm) to prepare an aqueous solution of glycol chitosan through which impurities were removed. Gold salt (HAuCl ₄ 3H ₂ O, 0.03 g) was dissolved in 100 ml of water to make a solution at a concentration of 1 mM, and then heated to 70 ° C. to prepare an aqueous gold salt solution. 100 ml of the gold salt solution was mixed with 300 ml of the aqueous solution of glycol chitosan and reacted for 24 hours in a stirrer to prepare gold nanoparticles coated with glycol chitosan.

Example 1 . Glycol  Blood clots using chitosan-coated gold nanoparticles monitoring  Biological CT video

1. After inducing thrombosis in mice CT  video

Mice (C57 / BL6) were inhaled anesthesia with 3% isoflurane and maintained at 37 ° C. using a Homeothermic blanket (Panlab). After placing the mouse on a surgical microscope (leica, EZ4, 8X), the incision was made to expose the left carotid artery (common carotid artery), and soaked for 5 minutes in 10% Ferric chloride solution (filter paper) 1 X 1 mm ² or 1 X 2 mm ² was placed in the carotid artery for 5 minutes. Thirty or 120 minutes after thrombosis was induced, 100 μl of glycol chitosan-coated gold nanoparticles (GC-AuNP) (0.9 mg / ml) were injected into the tail vein through a 1 cc syringe. After 5 minutes, images were acquired by micro CT (Nanofocusray, Polaris-G90, parameter: 65KV, 60uA, 512 X 512 reconstruction). In the acquired image, the location and size of the thrombus were observed in the image. In this case, a step of reconstructing the obtained Dicom file of the acquired image in 3D through Lucion software may be further performed.

Thrombosis is induced after carotid artery exposure in the mouse (left), and thus thrombosis is formed (right). FIG. 1 shows normal blood vessels (A) and blood clots that are tightly clogged after angiography. In B), the size and location of the thrombus are shown in FIG. 2. In addition, the CT images after inducing thrombosis in mice are shown in FIGS. 3 to 8.

As shown in Figure 3, 5 minutes after the gold nanoparticle injection was able to accurately image the location and size of the thrombus, rapid thrombus imaging was possible, it was confirmed that long-term monitoring was possible by tracking up to 48 hours.

As shown in FIG. 4, the biothrombogenic images in proportion to the histological thrombus size were confirmed. As shown in FIGS. 5 and 6, the thrombus size seen in the CT images according to the histological thrombus size (n = 35 per group). Was found to be proportional, and there was no change in the thrombus size seen in the time-phase CT images according to histological thrombus size (n = 5 per group). Therefore, it can be seen that quantitative thrombosis imaging is possible by using the gold nanoparticles coated with the glycol chitosan of the present invention as a CT contrast agent.

As shown in FIG. 7, since the gold nanoparticles injected once after the first thrombus was generated in the same experimental animal circulated for a long time, it was confirmed that recurrent thrombosis can be repeatedly diagnosed after the first thrombosis diagnosis.

As shown in FIG. 8, it was confirmed that a biological thrombus image proportional to histological thrombus size was obtained.

2. After thrombolytic agent administration to thrombosis-induced mice CT  video

In the thrombosis-induced mouse 1, a thrombolytic agent (Actilyse, Boehinger ingelheim Phama KG, tissue plasminogen activator, tPA 5mg / kg, 10mg / kg or 20mg / kg) is filled in a 1cc syringe, and a catheter is mounted and a drug injector ( Infusion was performed intravenously for 30 minutes using an infusion pump. Immediately after the injection, the images were re-acquired by micro CT to measure the blood clot solubility. The results are shown in FIGS. 9 to 12.

As shown in FIG. 9, it was confirmed that after the thrombolytic agent treatment, a portion of the thrombus moved to the distal blood vessel and the position was changed while the thrombus melted. Therefore, it can be seen that embolism diagnosis by thrombolytic therapy can be performed by using the gold nanoparticles coated with the glycol chitosan of the present invention as a CT contrast agent.

In addition, as shown in Figures 10 to 12, the thrombosis imaging technique according to the present invention reflects the thrombolytic treatment effect well, the faster the thrombolytic procedure is performed, the higher the thrombolytic capacity, the higher the thrombolytic system. As a result, it was confirmed that the thrombus volume was significantly decreased on the CT according to the thrombus lysis (n = 5 per group), and it was found that an appropriate reflection of the thrombus lysis monitoring effect was possible.

Claims (5)

1) injecting gold nanoparticles coated with glycol chitosan to animals other than humans with thrombosis, and
2) obtaining a CT image using a micro CT, CT-based thrombosis image monitoring method.
The method of claim 1, wherein the gold nanoparticles coated with glycol chitosan in step 1) are prepared by reacting an aqueous solution of glycol chitosan with an aqueous gold salt solution.
The method of claim 1, wherein the CT image further comprises a step of reconstructing in 3D through lucion software after step 2).
The method of claim 1, wherein the thrombosis is at least one selected from the group consisting of thrombosis, cardiovascular thrombosis and peripheral vascular thrombosis.
The method of claim 1, wherein the animal is at least one selected from the group consisting of dogs, mice, cats, monkeys, sheep, goats, cattle, horses, and pigs.

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