阅读说明：本技术 包括硅酸锰纳米颗粒的诊断成像肝组织特异性造影剂 (Diagnosing image liver tissue-specific contrast agent including manganous silicate nano particle ) 是由 李源宰 李贞姬 张文瑄 李寅洙 金晋究 于 2018-03-30 设计创作，主要内容包括：本发明涉及一种诊断成像的硅酸锰肝肿瘤特异性MRI造影剂以及使用MRI造影剂来表征肝组织的方法,该造影剂在酸性条件下释放锰离子(Mn<Sup>2+</Sup>)。在相对短的时间段内,根据正常肝组织和病变肝组织(尤其是肝肿瘤)中的诸如血管分布、细胞密度、线粒体活性、肝细胞亲和力等的组织特异性,T1加权成像显示出不同的图案,通过分析这样的T1加权成像,本发明允许在适当的时间检测肝肿瘤的疾病特异性特性。基于组织特异性,预期本发明非常有效地用于区分并诊断肝肿瘤的类型或者用于确定治疗效果,并且进一步有助于诊断除肝脏之外的器官中发生的疾病。(The method that hepatic tissue is characterized the present invention relates to a kind of manganous silicate liver tumour specificity MRI contrast agent of diagnosing image and using MRI contrast agent, the contrast agent discharge manganese ion (Mn in acid condition 2+ ).Within the relatively short period, according to the tissue specificity of vascular distribution, cell density, mitochondria activity, liver cell affinity in normal liver tissue and lesion hepatic tissue (especially liver tumour) etc., T1 weighted imaging shows different patterns, by analyzing such T1 weighted imaging, the present invention allows to detect the disease-specific properties of liver tumour in reasonable time.Based on tissue specificity, it is contemplated that the present invention is used very efficiently for distinguishing and diagnose the type of liver tumour or for determining therapeutic effect, and further helps in the disease for diagnosing and occurring in the organ in addition to liver.)

1. a kind of liver tissue-specific magnetic resonance imaging (MRI) contrast agent, the liver tissue-specific MRI contrast agent includes can The manganous silicate nano particle of manganese ion is discharged in acid condition, wherein manganese ion specifically accumulates in hepatic tissue, to produce The differentiable change pattern of specificity of raw MRI.

2. liver tissue-specific MRI contrast agent according to claim 1, wherein manganous silicate nano particle is absorbed into liver To discharge manganese ion in the Kupffer cell of tissue, and the manganese ion discharged is discharged to outside Kupffer cell and is specifically inhaled It receives and accumulates in hepatic tissue, to generate the variation specifically distinguished in hepatic tissue.

3. liver tissue-specific MRI contrast agent according to claim 2, wherein discharged from the outside of Kupffer cell The rate of release of manganese ion is such that manganese ion specifically accumulates in hepatic tissue, special in hepatic tissue to generate Distinguish to property.

4. liver tissue-specific MRI contrast agent according to claim 1, wherein contrast agent generates MRI variation at any time Pattern, MRI change pattern are distinguished in hepatic tissue by specificity.

5. liver tissue-specific MRI contrast agent according to claim 1, wherein it is close that contrast agent analyzes vascular distribution, cell Degree, mitochondria activity or liver cell affinity.

6. liver tissue-specific MRI contrast agent according to claim 3, wherein the rate of release of manganese ion passes through adjusting The porosity of nano particle and thickness degree control.

7. liver tissue-specific MRI contrast agent according to claim 1, wherein contrast agent is the hair for T1 weighted mri Bright contrast agent.

8. liver tissue-specific MRI contrast agent according to claim 1, wherein contrast agent is by liver tumour and normal hepatocytes group It knits and distinguishes.

9. liver tissue-specific MRI contrast agent according to claim 1, wherein contrast agent is by different types of liver tumour It distinguishes.

10. liver tissue-specific MRI contrast agent according to claim 1, wherein contrast agent monitoring liver tumour treatment is controlled Therapeutic effect.

11. liver tissue-specific MRI contrast agent according to claim 1, wherein manganous silicate nano particle has hollow knot Structure.

12. liver tissue-specific MRI contrast agent according to claim 1, wherein it includes oxygen that manganous silicate nano particle, which has, Change the structure of the core of manganese and the shell of silica.

13. liver tissue-specific MRI contrast agent according to claim 1, wherein the total weight based on nano particle, silicon Sour manganese nano particle includes manganese ion of the 0.5 weight % to 55 weight %.

14. liver tissue-specific MRI contrast agent according to claim 8, wherein it is right at any time in vivo that contrast agent generates Disease has the MRI variation of specificity.

15. liver tissue-specific MRI contrast agent according to claim 14, wherein MRI change pattern is brightness change.

16. liver tissue-specific MRI contrast agent according to claim 15, wherein obtained at any time after administration of contrast agents Between MRI change pattern have in normal liver tissue and gradually brighten and the gradually dimmed pattern after reaching maximum brightness.

17. tissue liver specificity MRI contrast agent according to claim 15, wherein hepatic tissue is hepatocellular carcinoma (HCC), And

The MRI change pattern of the Tissues of Hepatocellular Carcinoma obtained after administration of contrast agents at any time has such pattern: based on normal The MRI change pattern of hepatic tissue, hepatocellular carcinoma keeps dark state at any time after administration of contrast agents, and reaches in normal liver tissue Start to brighten when to maximum brightness, then keeps brightness, the MRI change pattern of the normal liver tissue gradually brightens and reaching It is gradually dimmed after to maximum brightness.

18. liver tissue-specific MRI contrast agent according to claim 15, wherein hepatic tissue is adenocarcinoma of colon (CAC), And

The MRI change pattern of the adenocarcinoma of colon obtained after administration of contrast agents at any time has such pattern: being based on normal hepatocytes group The MRI change pattern knitted, CAC is maintained at dark state at any time after administration of contrast agents, under state more darker than normal liver tissue by Gradual change is bright, or only has bright circle at edge, and the MRI change pattern of the normal liver tissue gradually brightens and reaching maximum It is gradually dimmed after brightness.

19. liver tissue-specific MRI contrast agent according to claim 15, wherein hepatic tissue is small intestine neuroendocrine Cancer (SNC), and

The MRI change pattern of the SNC obtained after administration of contrast agents at any time has such pattern: based on normal liver tissue MRI change pattern, SNC gradually brightens at any time and is maintained at the state brighter than normal liver tissue after administration of contrast agents, and Not dimmed, the MRI change pattern of the normal liver tissue gradually brightens and gradually dimmed when reaching maximum brightness.

20. a kind of method for characterizing hepatic tissue, the described method comprises the following steps: being applied according to claim 1 extremely to subject One or more of contrast agent described in 19；MRI is obtained by the hepatic tissue continuous imaging at any time to subject；And

Extract the time change pattern of the MRI of hepatic tissue.

21. the method for characterization hepatic tissue according to claim 20, wherein the MRI change pattern of liver at any time is MRI Brightness change pattern.

22. it is according to claim 20 characterization hepatic tissue method, wherein after administration of contrast agents by any time to by The hepatic tissue continuous imaging of examination person obtains in MRI, normal liver tissue have gradually brighten and when reaching maximum brightness by The dark pattern of gradual change.

23. the method for characterization hepatic tissue according to claim 20, wherein hepatic tissue is hepatocellular carcinoma (HCC), and

The MRI change pattern of the hepatocellular carcinoma obtained after administration of contrast agents at any time has such pattern: being based on normal hepatocytes group The MRI change pattern knitted, hepatocellular carcinoma is maintained at dark state at any time and then reaches in normal liver tissue after administration of contrast agents Start to brighten when maximum brightness, then keep brightness, the MRI change pattern of the normal liver tissue gradually brightens and reaching It is gradually dimmed after maximum brightness.

24. the method for characterization hepatic tissue according to claim 20, wherein hepatic tissue is adenocarcinoma of colon (CAC), and

The MRI change pattern of the adenocarcinoma of colon obtained after administration of contrast agents at any time has such pattern: being based on normal hepatocytes group The MRI change pattern knitted, CAC keeps dark state at any time after administration of contrast agents, under state more darker than normal liver tissue gradually It brightens, or only there is bright circle at edge, the MRI change pattern of the normal liver tissue gradually brightens and most light reaching It is gradually dimmed after degree.

25. the method for characterization hepatic tissue according to claim 20, wherein hepatic tissue is small intestine neuroendocrine carcinoma (SNC), and

The MRI change pattern of the SNC obtained after administration of contrast agents at any time has such pattern: based on normal liver tissue MRI change pattern, SNC gradually brightens at any time and is maintained at the state brighter than normal liver tissue after administration of contrast agents, and Not dimmed, the MRI change pattern of the normal liver tissue gradually brightens and gradually dimmed when reaching maximum brightness.

26. a kind of for providing the method for the information determined about the diagnosis of liver tumour and the type of liver tumour, the method packet It includes following steps: subject is applied according to claim 1 to one or more of contrast agent described in 19；And

MRI is obtained by the hepatic tissue continuous imaging at any time to subject.

Technical field

The present invention relates to a kind of contrast agent for hepatic tissue imaging diagnosis, are included in acidity more particularly, to one kind Under the conditions of discharge manganese ion (Mn²⁺) manganous silicate the contrast agent for liver cancer-specific imaging diagnosis, or more specifically, relate to And a kind of MRI (magnetic resonance imaging) contrast agent for liver tumour specificity imaging diagnosis.

Background technique

Effect of the contrast agent (contrast agent) in magnetic resonance imaging (MRI) is for by enhancing to lesion Detectability or characterize lesion using organ or the radiography of lesion enhancing (contrast enhancement) to improve The ability of diagnosis or antidiastole.Currently, because paramagnetism Gd³⁺Chelates contrast agent is a kind of difference based on vascular distribution Non-specific cell external solution (extracellular fluid, ECF) contrast agent, so paramagnetism Gd³⁺Chelates contrast agent It is widely used in liver MRI.Among them, the referred to as Gd-EOB- of liver specificity (liver-specific) contrast agent DTPA is only absorbed in liver cell, and there is hepatobiliary excretion object to be discharged in biliary ductal tree.However, the MR using ECF is imaged Need the hard situation of such as rapid image acquisition and precise start time.In addition, by the expected Gd occurred due to removing chelating³⁺From A possibility that kidney source property caused by son is systemic fibrosing.In addition, having developed such as SPIO (superparamagnetic Iron oxide, Superparamagnetic Iron Oxide) and MnDPDP (mangafodipir trisodium, mangafodipir trisodium) radiography Agent is to overcome the problems, such as ECF, but since them are rarely employed in current various problems.

SPIO is the nano particle preferentially absorbed by Kupffer cell (Kupffer cell), and Kupffer cell is to constitute liver One of dirty cell.In terms of MRI imaging, because SPIO only reduces the T2 weighted graph in the normal liver including Kupffer cell The signal strength of picture, so SPIO is used as T2 contrast agent.The absorbed aspect in Kupffer cell, SPIO can be liver spy Anisotropic contrast agent.However, SPIO has the characteristic to liver tumour without identification because SPIO does not influence the signal strength of liver cancer The critical defect of diagnosis.

The MnDPDP of intravenous administration passes through the Zn in blood flow²⁺Ion is migrated by metal, and discharges free Mn²⁺Ion.Blood Mn in stream²⁺Ion increases T1 signal strength by the various absorbed organs including liver, and to t1 weighted image.Therefore, MnDPDP is T1 contrast agent.The MnDPDP absorbed by liver is absorbed by liver cell and liver tumour simultaneously, but MnDPDP is swollen according to liver The characteristic of tumor can seem whiter (high signal intensity), darker (low signal intensity).Since MnDPDP is also inhaled in liver cell It receives, therefore is not easy to distinguish between liver cell and liver tumour, cause gradually to be not used because detecting liver cancer the problem of.It changes Yan Zhi, MnDPDP, which have, to be absorbed into liver cell and is discharged into the liver and gallbladder secretion in bile duct.However, MnDPDP is also owned The cell of organ and liver cell absorb, and are not studied sufficiently this.Therefore, MnDPDP is known as liver specificity Contrast agent has limitation.

Currently used Gd³⁺Class contrast agent has high-precision in terms of the detection of liver cancer and characterization, but such as discharge Gd³⁺The problem of the clinical toxicity (for example, kidney source property is systemic fibrosing) of ion and environmental pollution etc. is occurring.It can be right Gd³⁺The Mn that class contrast agent is supplemented²⁺Class contrast agent (MnDPDP) has been safe due to hypotoxicity, but in basis There is limitation to distinguish various liver cancers in the characteristic of cancer.

The problem of despite the presence of above mentioned MnDPDP, but the present inventors have realized that and Gd³⁺Class contrast agent is compared The demand of the lesser T1 contrast agent of toxicity simultaneously develops (referring to 10-2016-0023963 Korean Patent public affairs it It opens).However, due to the Mn discharged from Kupffer cell²⁺Speed is slow, therefore it has acquisition liver cancer image relatively long Disadvantage, and not yet study a possibility that antidiastole is used for according to the characteristic of liver cancer.

Therefore, antidiastole must be carried out according to cancer characteristic for the MRI contrast agent of imaging diagnosis.There is an urgent need to develop It is capable of the liver cancer-specific MRI contrast agent of image of the quick obtaining with this antidiastole characteristic.

Summary of the invention

[technical problem]

The present inventor specifically has found the Mn of manganous silicate nano particle²⁺The release of ion and relaxation properties and T1 weighted graph Picture, and the present invention is completed based on following content: release manganese ion (Mn²⁺) manganous silicate nano particle (such as, hollow silicic acid Manganese (HMS) nano particle) or the structure of manganese oxide core and silica shell there is hypotoxicity, quick contrasting effects and can It is distinguished between liver cancer type by the pattern of different t1 weighted images.

Specifically, the MnO core in nano particle including manganese oxide core and silica shell is at low ph condition (pH 4-5) Under it is easy to dissolve, it is special as reactivity and liver and since the cellular conditions of Kupffer cell are in pH condition Property MRI contrast agent is useful.By discharging Mn in acid condition²⁺Ion, contrast agent can be enhanced positioned at normal liver tissue In Kupffer cell to the radiography of cancerous lesion.

In the previous research of the present inventor, Mn²⁺The nano SiO 2 particle of doping is used as contrast agent.As this hair A kind of hollow manganous silicate (HMS) of bright manganous silicate nano particle discharges free Mn at a low ph²⁺, but manganous silicate nanometer Structure control of the rate of release of grain by nano particle, and the Mn of hollow manganous silicate (HMS)²⁺Rate of release ratio Mn²⁺Doping Nano SiO 2 particle is faster, so that the different T1 that the early stage after being injected intravenously contrast agent shows liver add Weigh MRI imaging.

It, can be by adjusting nano particle with manganese oxide core-silica shell structure nano particle The thickness of middle silica shell and/or porosity control Mn²⁺Release, can pass through adjust Mn²⁺Release to outside particle comes Control radiography Enhanced time.

The difference of the radiography enhancing pattern of T1 weighted mri can provide valuable diagnosis letter for the pathological characters of liver tumour Breath, and distinguish primary malignant tumor and metastatic tumo(u)r and liver tumour type.It has been found by the present inventors that by low in adjusting Mn under the conditions of pH²⁺A series of pH reactivity MnO nano SiO 2 particles are prepared in the case where the rate of release and based on disease Reason feature and angiogenesis measure the various change of T1 weighted mri between different tumor models, it is determined that for lesion detection and Characterize the optimization rate of release of the NP (nano particle) of design.

Hereinafter, it will be described in detail the present invention.

The present invention relates to a kind of MRI contrast agent for hepatic tissue, which includes to release in acid condition Put the manganous silicate nano particle of manganese ion, wherein manganese ion specifically accumulates in hepatic tissue, can distinguish to generate specificity MRI change pattern.

Manganous silicate nano particle is absorbed into the Kupffer cell of hepatic tissue to discharge manganese ion, and the manganese discharged from Son is discharged to outside Kupffer cell and is specifically absorbed and accumulated in hepatic tissue, is become with generating the differentiable MRI of specificity Change pattern.

The rate of release of the manganese ion discharged from Kupffer cell can make manganese ion specifically accumulate in hepatic tissue In and have in hepatic tissue generate specificity distinguish MRI change pattern rate of release.

The present invention provides a kind of liver specificity MRI contrast agent, which includes being capable of specificity Distinguish the manganous silicate nano particle of hepatic tissue (specifically, the type of liver cancer and origin) and liver cancer and normal liver tissue in ground.

It has been found by the present inventors that being loaded with to distinguish the type of liver cancer and origin by the manganese of endocytosis to Kupffer cell The rate of release and/or burst size of manganese ion in the nano particle of ion are important.Specifically, nano particle must moment Discharge excessive manganese ion (Mn²⁺), specifically to distinguish hepatic tissue.

Contrast agent comprising manganous silicate nano particle of the invention can specifically be accumulated in hepatic tissue with manganese ion Rate of release discharge manganese ion, specifically to generate MRI change pattern in hepatic tissue.

It is possible in accordance with a preferred embodiment of the present invention to control manganese ion by the porosity and thickness of adjusting nano particle Rate of release.

According to one embodiment of present invention, liver specificity MRI contrast agent can analyze vascular distribution, cell density, Mitochondria activity or liver cell affinity.

According to an embodiment of the invention, MRI contrast agent can be the T1 contrast agent with radiography enhancing T1 weighting type.

In addition, when a contrast agent is employed, the type of liver cancer and normal liver tissue and liver cancer can be distinguished, and monitor liver cancer Therapeutic effect.

Parenchymal tissue, and the class of liver cancer can be can be by using the normal liver tissue that contrast agent of the invention is distinguished Type may include the liver cancer of commonly known all kinds.Specifically, liver cancer may include the benign cancer of liver, by liver cell Metastatic hepatocellular carcinoma caused by primary carcinoma of liver caused by the canceration of cancer or the organ in addition to liver.Based on referring to blood Pipe, benign tumour includes hemangioma, adenoma of liver and Focal nodular hyperplasia (FNH) etc., and primary carcinoma of liver includes hepatocellular carcinoma (HCC), cholangiocarcinoma, hepatoblastoma and angiosarcoma etc., metastatic hepatic carcinoma include dividing in adenocarcinoma of colon (CAC) and small enteric nervous Secrete cancer (SNC).

Preferred embodiment in accordance with the present invention, manganous silicate nano particle can have hollow structure.

In the present invention, " hollow manganous silicate (HMS) nano particle " has high Mn content, and such as swallows when being exposed to When cell-inner body environment acid condition, since the amorphous property of manganese can interrupt and with the release of a large amount of manganese ions.Therefore, Manganous silicate nano particle is easy to filter from blood and preferentially absorbed by Kupffer cell, then a large amount of Mn²⁺Ion is general from library Not cell discharges.The Mn of release²⁺Ion is according to the vascular distribution of cancer, cell density, mitochondria activity or liver cell affinity Equal distribution is in various hepatic tissues.For example, the Mn of release²⁺Ion is absorbed and accumulates in cancer, therefore nano particle has group Knit specificity.

According to an embodiment of the invention, manganous silicate nano particle can have manganese oxide core-silica shell structure.

Hereafter, have manganese oxide core-silica shell structure nano particle by MnO SiO₂It indicates.Silica shell is protected Protect Mn²⁺Releasing immediately in low ph conditions, and improve the dispersion of particle under conditions of neutral ph.

Mn can be controlled by adjusting thickness and the aperture of silica shell²⁺Rate of release.With silica shell Thickness increase, rate of release reduces, and as porosity increases, rate of release increases.

The present invention relates to it is a kind of by control manganese oxide core-silica shell nanoparticles silica shell thickness and Aperture is come the method that efficiently differentiates liver cancer type.For example, being capable of providing the nano particle with optimum structure to discharge Mn²⁺, So that the difference of MRI image change pattern at any time between liver tumour tissue and normal liver tissue is apparent from or make can To distinguish liver cancer in a short time.

The thickness of manganese oxide core-silica shell nanoparticles silica shell is preferably 0.5nm to 20nm or 1nm To 15nm, or more preferably 2nm to 12nm.When thickness is more than the upper limit of thickness range, there is significantly reduced Mn²⁺It releases The shortcomings that putting rate and long MRI time of measuring.When thickness is less than lower thickness, Mn²⁺Excessively release rapidly, so that being difficult to Distinguish the type of liver cancer.

Example 11 according to the present invention, it can be seen that the difference of MRI change pattern at any time be according to the type of liver cancer and The time of each type of nano particle and generate, and in a short period of time only by using with suitable structure, inspection The suitable nanoparticle contrast agent with liver tumour type is surveyed, liver cancer and liver tumour class can be efficiently differentiated in a short time Type.

Preferred embodiment in accordance with the present invention, in manganous silicate nano particle of the invention comprising 0.5wt% to 55wt% or The manganese ion of the amount of 15wt% to 55wt% or more preferably 19wt%-55wt% or 20wt%-47wt%.When the content When manganese ion includes in nano particle, the radiography of MRI contrast agent enhances and is improved.

Preferred embodiment in accordance with the present invention, MRI contrast agent are that pH response signal is enhanced, this is that maximum of the invention is special One of sign.

It is highly preferred that hollow manganous silicate (HMS) MRI contrast agent has 0.12s^-1·mM^-1R₁Value, which is external neutrality Under the conditions of specific relaxivity.This indicates the 7.8s with free manganese ion^-1·mM^-1R₁Value is compared, by not reducing water proton Relaxation time, do not influence the signal strength of MR image.On the other hand, Mn²⁺Ion increases in acid condition from the release of HMS Add, the radiography enhancing for causing MRI to be imaged.According to preferred embodiment, acid condition is pH 2-5.5, or more preferably pH 3-5。

MRI can measure T1 and T2 image by the nuclear spin relaxation of hydrogen molecule in measurement hydrone.MRI contrast agent quilt It is divided into T1 contrast agent and T2 contrast agent, and for amplifying T1 or T2 signal.Each of T1 and T2 respectively indicate MRI center certainly Spin-lattice relaxation time or spin spin relaxation time after rotation excitation, generate different contrasting effects.

Preferred embodiment in accordance with the present invention, MRI contrast agent of the invention are T1 radiography Contrast-enhanced MRI contrast agent.Generally, Compared with water, the manganese ion of the paramagnet in MRI contrast agent of the invention shows bright or positive contrasting effects.

The MRI disease specific change pattern that contrast agent of the invention changes over time under the conditions of can produce in vivo.

Term " specificity " in the present invention indicates MRI contrast agent of the invention according to internal certain organs or the spy of tissue Property with relatively large amount accumulation or release.Disease type of the invention is not particularly limited, and can be in organ All diseases, it is therefore preferable to liver diseases.

For example, MRI contrast agent of the invention has liver specificity, i.e., they by existing only in normal liver tissue first In Kupffer cell be absorbed in the form of nano particle and largely existed in liver.With Mn²⁺It is thin that form is absorbed into liver The process of born of the same parents can not be said to be liver specificity, but being discharged to the process in bile duct is liver specificity.According to the above, when When liver tumour occurs, whether MRI contrast agent of the invention can have liver tumour specificity to apply highly usefully according to tumour In diagnosing tumour state and tumor type.

According to another embodiment of the present invention, MRI contrast agent causes disease specific signals to enhance in condition in vivo.

The enhancing of disease specific radiography is also one of maximum feature of the invention, and is indicated in the injection MRI contrast agent phase Between, tumor region or borderline tumor characteristic (vascular distribution, cell density, mitochondria activity, liver cell based on liver tumour type Affinity etc.) show that difference radiography enhances.These characteristics can be used for that the type for distinguishing liver tumour or true is imaged by MR Determine the therapeutic effect of liver tumour.

In one embodiment of the invention, as preparing HMS and check the manganese releasability and T1 contrasting effects of HMS As a result, it is possible to all Mn of release²⁺It releases immediately in acid condition, and makes free Mn in 15 minutes²⁺The concentration of ion It maximizes (see example 2).In another example, according to the liver tumour of three types (HCC- primary, SNC, CAC- metastatic) To distinguish t1 weighted image (see example 4).

In another embodiment of the invention, preparation has manganese core-silica shell structure nano particle (MnO SiO₂), and thickness and/or the aperture of silica shell are controlled to prepare the [email protected] of three types₂(HCC- primary, SNC With CAC- metastatic).When being injected intravenously every kind of nanoparticle contrast agent, different t1 weighted images is obtained.

Therefore, nano particle of the invention shows the t1 weighted image of different pattern according to the characteristic of liver tumour.Specifically Ground, nano particle of the invention can be used for diagnosing and distinguishing the type of liver tumour, or be applied to determine liver tumour treatment effect Various purposes and purposes needed for fruit.

According to an embodiment of the invention, the MRI mobile image obtained after applying the MRI contrast agent for hepatic tissue Change pattern can be the brightness change pattern of MRI image.

When listing along the MRI image of time shooting, brightness change pattern can be judged at any time.

The radiography enhancing change pattern of the MRI image obtained after administration of contrast agents at any time, which may be such that, to be applied Gradually blast and become in chronological order farthest with the radiography enhancing change pattern of normal liver tissue after contrast agent It is dimmed after bright.Particularly, normal liver tissue can be parenchymal tissue.

According to an embodiment of the invention, hepatic tissue can be hepatocellular carcinoma (HCC).In the normal tissue by contrast agent application The MRI obtained later changes with time in pattern, and the brightness of MRI is gradually increased as time goes by after administration of contrast agents And reach maximum brightness, it is then dimmed.Based on the change pattern of the MRI in normal liver tissue, hepatocellular carcinoma can have this The change pattern of the MRI of sample: Tissues of Hepatocellular Carcinoma keeps dark state over time after administration of contrast agents, from normal hepatocytes group Tissue region starts to brighten when reaching maximum brightness, and keeps bright state.

According to an embodiment of the invention, the part for being expected to hepatocellular carcinoma shows the change pattern of such MRI: in quilt HMS injection after, radiography unevenly enhances immediately from tumour peripheral portion, then uniformly enhancing with fill central part until 24 hours.Expected part can be determined as hepatocellular carcinoma.

According to one embodiment of present invention, in the case where hepatic tissue is adenocarcinoma of colon (CAC), it is based on normal liver tissue In MRI change pattern, adenocarcinoma of colon tissue keeps black state immediately or compares normal liver tissue after administration of contrast agents The darker state of MRI brightness, then only gradually brighten in peripheral portion or become bright circle, in normal liver tissue The change pattern of MRI is such that after administration of contrast agents, the brightness of MRI is gradually increased as time goes by and reaches most Big brightness, it is then dimmed.

According to an embodiment of the invention, the initial stage after part for being detected as liver tumour is kept from injection HMS To 24 hours when the radiography enhancing of completely opposite reduction, specificity portion can be determined that CAC.

According to one embodiment of present invention, in the case where hepatic tissue is small intestine neuroendocrine carcinoma (SNC), SNC is aobvious The MRI obtained after applying HMS is shown to change with time pattern, in the change pattern, based in normal liver tissue The change pattern of MRI, the brightness of MRI is gradually increased as time goes by after administration of contrast agents, and is maintained at than normal The brighter state of hepatic tissue without dimmed, the change pattern of the MRI in normal liver tissue be such that administration of contrast agents it Afterwards, the brightness of MRI is gradually increased as time goes by and reaches maximum brightness, then dimmed.

According to an embodiment of the invention, the initial stage after part for being detected as liver tumour is kept from injection HMS When to 24 hours, completely opposite holding height radiography enhanced, specificity portion can be determined that SNC.

According to one embodiment of present invention, by by [email protected]₂Nano particle be injected into every kind of cancer (HCC, CAC, SNC it in) and measures as the MRI of time course changes, can detecte and distinguish liver cancer.Although the change pattern of MRI according to [email protected]₂The thickness of the shell and porosity of nano particle occur in being spaced in different times, but the change pattern of brightness and darkness It is the common features of cancer.

The HCC of example 9 according to the present invention, in intravenous administration [email protected]₂The MRI variation obtained after contrast agent In pattern, wherein the enhancing of high radiography is kept in 30 minutes to 1 hour and the enhancing of low radiography is kept in 1 hour to 24 hours Tissue part can be determined that normal liver tissue.It is dark (black) when the part is interior when 30 minutes small to 4 or only exists Show that radiography enhances in neighboring area, when then showing the enhancing of high radiography in 4 hours to 24 hours, which can be by It is determined as HCC tissue.

Wherein high radiography enhancing is kept in 30 minutes to 1 hour and the enhancing of low radiography is protected in 1 hour to 24 hours The tissue part held can be determined that normal liver tissue.

In [email protected]₂In the case where as contrast agent, in intravenous administration [email protected]₂It is taken after contrast agent In the MRI change pattern obtained, show that high radiography enhances when the part is interior when 45 minutes are small to 4, it is then small at 4 hours to 24 When it is interior show gradually decrease radiography enhancing when, which can be determined that normal liver tissue.In the MRI of normal liver tissue Change pattern in, when the part has such change pattern: the part is dark (black in 45 minutes to 4 hours Color), it is then gradually diffused into tumour in the enhancing of 4 hours to 24 hours interimages from the outside of height enhancing, which can be by It is determined as HCC tissue.

In [email protected]₂In the case where as contrast agent, in intravenous administration [email protected]₂It is obtained after contrast agent MRI change pattern in, show the enhancing of high radiography when the part is interior when 45 minutes are small to 8, then radiography enhancing was at 8 hours When gradually decreasing in 24 hours, which can be determined that normal liver tissue.In the change pattern of the MRI of normal liver tissue In, when the part has such change pattern: the part is that dark (black) or radiography increase in 45 minutes to 8 hours It is shown in neighboring area by force, then radiography enhancing is high, the part in entire tumor region in 8 hours to 24 hours It can be determined that HCC is organized.

The CAC (HT29) of example 11 according to the present invention, in intravenous administration [email protected]₂It is obtained after contrast agent In MRI change pattern, when the part shows high radiography enhancing in 15 minutes to 45 minutes, then at 1 hour to 24 hours When showing the radiography enhancing gradually decreased later, which can be determined that normal liver tissue.In the MRI of normal liver tissue Change pattern in, be black or when being only bright in neighboring area, the part when the part is interior when 15 minutes are small to 24 It can be determined that CAC is organized.

In intravenous injection [email protected]₂In the MRI change pattern obtained after contrast agent, when the part was at 30 minutes High radiography enhancing is shown in 4 hours, when then showing the radiography enhancing gradually decreased after 4 hours to 24 hours, The part can be determined that normal liver tissue.In the change pattern of the MRI of normal liver tissue, when the part at 30 minutes extremely It is black in 24 hours or when being only bright in neighboring area, which can be determined that CAC is organized.

In intravenous injection [email protected]₂In the MRI change pattern obtained after contrast agent, when the part was at 15 minutes High radiography enhancing is shown in 4 hours, when then showing the radiography enhancing gradually decreased after 8 hours to 24 hours, The part can be determined that normal liver tissue.In the change pattern of the MRI of normal liver tissue, when the part at 15 minutes extremely Be in 4 hours it is black or only in neighboring area be it is bright, then only in the frontier district of tumour after 8 hours to 24 hours When showing radiography enhancing in domain, which can be determined that CAC is organized.

The SNC (STC-1) of example 12 according to the present invention, in intravenous injection contrast agent [email protected]₂、[email protected] pSiO₂With [email protected]₂In every kind after obtain MRI change pattern in, two parts at 15 minutes to 1 hour It is interior to be distinguished, but in 4 hours to 24 hours the latter partially due to gradually radiography enhance and in the case where brighten, this One part can be determined that SNC is organized.

Preferred embodiment in accordance with the present invention, MRI contrast agent cause internal signal strength to enhance with continuous time sequencing. More specifically, being shown in normal liver tissue to 5 hours within 0.1 hour after injecting in vivo when applying HMS contrast agent High signal intensity, and in vivo high signal intensity was shown in liver diseases tissue to 24 hours within 6 hours after injection.

The characteristic of this time difference signal enhancing is one of maximum feature of the invention, because by the library in normal liver tissue The HMS that kupffer cell absorbs releases Mn in acidic endosomes environment²⁺Ion, with after injecting HMS contrast agent until 5 hours High signal intensity is shown in normal liver tissue, but shows low signal intensity in hepatic disease tissue.Then, because from The Mn for discharging and spreading outside Kupffer cell²⁺Ion is by hepatic disease tissue resorption, so showing in normal liver tissue low Signal strength, but high signal intensity is shown in hepatic disease.

In other words, normal liver tissue whiten in t1 weighted image then blackening, but hepatic disease tissue black then become It is white.

Due to the signal enhancing of this time difference, can be received in a series of single MRI pictures with the mode of reversed radiography Collection clearly illustrates double MRI pictures of hepatic disease tissue, and can increase the recall rate of hepatic disease tissue and identification is examined A possibility that disconnected.

The embodiment of the present invention is to provide a kind of method for characterizing hepatic tissue, method includes the following steps: to tested Person applies at least one contrast agent；Obtain by with the time to the hepatic tissue continuous imaging of subject and the magnetic resonance figure that obtains Picture；And extract the time change pattern of the magnetic resonance imaging of hepatic tissue.

Because hepatic tissue characteristic caused by the magnetic resonance imaging of hepatic tissue change with time the above-mentioned of pattern and contrast agent It is identical described in part.

In addition, the embodiment of the present invention provides a kind of information for providing and determining about Diagnosis of Hepatic Tumors and liver tumour type Method, method includes the following steps: applying at least one contrast agent to subject；It obtains by as the time is to subject's Hepatic tissue continuous imaging and the magnetic resonance image obtained.

The information determined about Diagnosis of Hepatic Tumors and liver tumour type can be the brightness change of magnetic resonance imaging at any time Pattern.

Subject can be animal, it is therefore preferable to mammal, and can not include people.

[The effect of invention]

The present invention relates to a kind of MRI contrast agent for hepatic tissue, which includes discharging manganese in acid condition Ion (Mn²⁺) manganous silicate nano particle.The present invention relates to a kind of MRI contrast agents for hepatic tissue, which can Inhibit vascular distribution, cell density.The pattern of t1 weighted image is according to tissue characteristics (such as vascular distribution, cell of liver tumour Density, mitochondria activity, liver cell affinity etc.) it is different.By analyzing t1 weighted image, can detect in a short time The characteristic of liver tumour, it is therefore contemplated that for distinguishing the type of liver tumour and determining that therapeutic effect is highly useful.

Detailed description of the invention

Fig. 1 shows the manganese releasability and T1 contrast enhancing effects of hollow manganous silicate (HMS) nano particle, a) synthesizes The TEM image of nano particle, b) pH 5 citric acid (salt) buffer suspension TEM image, c) from HMS particle discharge Mn²⁺'s Amount and d) t1 weighted image.

Fig. 2 is t1 weighted image and function machine after by HMS (3mg/kg) intravenous injection into hepatocellular carcinoma (HCC) model The schematic diagram of system.

Fig. 3 a shows liver tumour (the primary HCC, metastatic SNC (small of representative three types Intestinal neuroendocrine carcinoma, small intestine neuroendocrine carcinoma) and CAC (colonic Adenocarcinoma, adenocarcinoma of colon)) t1 weighted image, specifically, a) image enhancement of HCC model, b) SNC model Image enhancement, c) CAC model image image enhancement, Fig. 3 b shows detailed picture.

Fig. 4 is shown by according to anti-prohibin antibody (or being inhibin/tumor suppressor antibody) dyeing Make mitochondria dye the cell of (brown) distribution and activity and by according to hematoxylin dyeing make nuclear targeting (it is blue Color) liver tumour type cell density and vesica distribution.

Fig. 5 is the radiography enhancing effect shown by FAST SPIN echo t1 weighted image to the vascular distribution of three kinds of liver tumours The figure of fruit.

Fig. 6 be show using NADH- tetrazolium () reduction enzyme dyeing hepatic artery ligation after in HAO (arteria hepatica closes Plug) (on) and HCC necrosis (under) at acquisition t1 weighted image figure.

Fig. 7 is [email protected]₂(MnO-SiO₂- 0.5hr) nano particle, [email protected]₂(MnO-SiO₂-2hr) Nano particle, [email protected]₂(MnO-SiO₂- 12hr) schematic diagram of nano particle, micro-image and for synthesizing [email protected] 5nm-SiO₂(MnO-SiO₂- 0.5hr) nano particle, [email protected]₂(MnO-SiO₂- 2hr) nano particle, [email protected] SiO₂(MnO-SiO₂- 12hr) nano particle ingredient and its dosage.

Fig. 8 a is that [email protected] is shown respectively₂(MnO-SiO₂- 0.5hr) nano particle, [email protected]₂(MnO- SiO₂- 2hr) nano particle, [email protected]₂(MnO-SiO₂- 12hr) nano particle perspective view figure and according to MnO- SiO₂-0.5h、MnO-SiO₂- 2h and MnO-SiO₂The sequence Mn of -12h²⁺Increase rate of release figure.

Fig. 8 b is to show Mn of passing at any time under conditions of pH 5, pH 6,7 pH²⁺Ion is from [email protected]₂ (MnO-SiO₂- 0.5hr) rate of release figure.

Fig. 8 c is to show Mn of passing at any time under conditions of pH 5, pH 6,7 pH²⁺Ion is from [email protected]₂ (MnO-SiO₂- 2hr) rate of release figure.

Fig. 8 d is to show Mn of passing at any time under conditions of pH 5, pH 6,7 pH²⁺Ion is from [email protected]₂ (MnO-SiO₂- 0.5hr) rate of release figure.

Fig. 8 e is shown for [email protected]₂(MnO-SiO₂-0.5hr)、[email protected]₂(MnO-SiO₂- 2hr)、[email protected]₂(MnO-SiO₂- 0.5hr) Mn²⁺The figure of the comparison of the rate of release of ion.

Fig. 9 a is shown by from [email protected]₂(MnO-SiO₂- 0.5hr) nano particle, [email protected]₂ (MnO-SiO₂- 2hr) nano particle, [email protected]₂(MnO-SiO₂- 0.5hr) nano particle release Mn²⁺What ion obtained The figure of the contrast intensity variation of t1 weighted image.

Fig. 9 b is the figure for showing the numerical value by quantifying the contrast intensity of the image shot in Fig. 9 a.(R₁, unit= Second).

Fig. 9 c is to show Mn²⁺The concentration of ion and the contrast intensity (R of image₁) between relationship figure.

Figure 10 a shows the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 0.5hr) nanometer Particle intravenous injection is into HCC tumor model 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, 24 hours later The MRI of shooting.

Figure 10 b shows the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 2hr) nanometer Particle intravenous injection is into HCC tumor model 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, 24 hours later The MRI of shooting.

Figure 10 c shows the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 12hr) nanometer Particle intravenous injection is into HCC tumor model 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, 24 hours later The MRI of shooting.

Figure 11 a to Figure 11 e is shown by [email protected]₂(MnO-SiO₂- 12hr) nano particle, [email protected] SiO₂(MnO-SiO₂- 2hr) nano particle, [email protected]₂(MnO-SiO₂- 0.5hr) nano particle is injected into each organ Later, the figure of the result of SNR (small noise ratio) value for each Organ size contrast enhancing effects at any time: Figure 11 a is directed to Liver tumour, Figure 11 b are directed to normal liver tissue, and Figure 11 c is directed to kidney, and Figure 11 d is directed to spleen, and Figure 11 e is directed to intestines.

Figure 12 a is the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 0.5hr) nano particle It claps within 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, 24 hours after being injected intravenously in CAC tumor model The MRI taken the photograph.

Figure 12 b shows the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 2hr) nanometer Particle intravenous injection into CAC tumor model after 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, it is 24 small When the MRI that shoots.

Figure 12 c shows the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 12hr) nanometer Particle intravenous injection into CAC tumor model after 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, it is 24 small When the MRI that shoots.

Figure 13 a is the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 0.5hr) nano particle It claps within 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, 24 hours after being injected intravenously in SNC tumor model The MRI taken the photograph.

Figure 13 b shows the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 2hr) nanometer Particle intravenous injection into SNC tumor model after 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, it is 24 small When the MRI that shoots.

Figure 13 c shows the MRI shot before injection and by [email protected]₂(MnO-SiO₂- 12hr) nanometer Particle intravenous injection into SNC tumor model after 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours, it is 24 small When the MRI that shoots.

Figure 14 a shows the MRI contrastographic picture collected by Figure 10 a to Figure 10 c.

Figure 14 b shows the MRI contrastographic picture collected by Figure 12 a to Figure 12 c.

Figure 14 c shows the MRI contrastographic picture collected by Figure 13 a to Figure 13 c.

Figure 15 a is shown by [email protected]₂(MnO-SiO₂- 0.5hr) contrast agent is administered in three kinds of tumor models Every kind after the variation passed at any time of MRI contrastographic picture.

Figure 15 b is shown by [email protected]₂(MnO-SiO₂- 2hr) contrast agent is administered in three kinds of tumor models The variation that MRI contrastographic picture is passed at any time after every kind.

Figure 15 c is shown by [email protected]₂(MnO-SiO₂- 12hr) contrast agent is administered in three kinds of tumor models Every kind after the variation passed at any time of MRI contrastographic picture.

Specific embodiment

Hereinafter, the preferred embodiment of the present invention will be described in order to understand the present invention.However, providing following example Merely to it is easier to understand the purpose of the present invention, and the present invention is not limited by following example.

[example]

The preparation and step of example 1, material

1-1, experimental material

Use the MnCl of no any purifying such as purchase₂·4H₂O (kanto), enuatrol (TCI), 1- octadecene (Aldrich)、CO-520 (Aldrich), tetraethyl orthosilicate (Acros), NH₄OH(Samchun Chem.)、Ni (NO₃)6H₂O(Strem)、Cu(NO₃)₂·3H₂O(Strem)、NaBH₄(Samchun Chem.), 2- [methoxyl group (polyethylene oxygen Base)-propyl] 9-12- trimethoxy silane (MPEOPS, Gelest, Inc.), fluorescein isothiocynate (FITC, Aldrich) with And 3-aminopropyltriethoxysilane (APTES, Aldrich).

The preparation of 1-2, HMS (hollow manganous silicate)

According to the technology being previously reported, by making [email protected]₂/Cu²⁺It is (hollow to anneal and then be etched selectively to [email protected] The NP of structure) external silica shell (Kim, J.G., Kim, S.M.&Lee, I.S.Mechanistic insight into the [email protected] transformation of [email protected] nanospheres incorporating Ni²⁺ions Toward a colloidal hollow nanoreactor.Small 11,1930-1938 (2015)) Lai Hecheng HMS is (hollow Manganous silicate) nano particle (NP).The HMS of synthesis has following property；It include that the Cu encapsulated receives in hollow hole (14 ± 2nm) The HMS (39 ± 2nm) of meter Jing Ti (9 ± 1nm) is according to solid solution-hollow (solid-state-hollowing) from comprising combining Cu²⁺SiO₂The nanocrystal synthesis of nanosphere.

The release and relaxation properties of 1-3, HMS

At room temperature, use 8mg's in citric acid (salt) buffer and distilled water of 16mL and pH 5 and pH 6.2 HMS suspension quantifies manganese ion from the release profiles of HMS.At 15 minutes, 1 hour, 7 hours and 20 hours from sample Product collect every kind of HMS suspension of 4mL, and the nano particle (NP) for removing sample by being centrifuged 10 minutes with 15,000rpm, And every kind of supernatant is filtered by using the injecting type filter that aperture is 0.2mm.Use ICP-AES and 3.0-T clinic MR Scanner (Philip, Achieva version 1.2, Philips Medical systems, most preferably, and Holland) every kind in every kind of supernatant is released The amount for the manganese ion put and the T1 relaxation time of proton are quantified.

The preparation (primary HCC, metastatic SNC and CAC) of 1-4, animal model

Buying 6 week old BALB/C nude mices from Orient Bio (Soul, South Korea (Seoul, Korea)), (HCC model is male And metastatic carcinoma model is female).All zooscopies all pass through Samsung Life Science Institute Animal Lab. and use committee Member's meeting (Animal Laboratory Use Committee of the Samsung Life Sciences Institute) Approval.User HCC cell strain (HepG2, Korea Cell strain library (Korean Cell Line Bank)) constructs HCC model, User CAC cell strain (HT29, ATCC) and mouse SNC cell strain (STC-1, ATCC) construct metastatic carcinoma model.It will be thin Born of the same parents' strain is maintained in DMEM culture medium (Dulbecco's Modified Eagle's medium, STC-1), the DMEM culture medium Comprising minimum essential medium (HepG2), McCoy 5a culture medium (HT29) and 10% fetal calf serum (Invitrogen) and 1% antibiotic (ThermoFisher).By cell in 5%CO at 37 DEG C₂Middle culture and with 0.25% trypsase/EDTA (ThermoFisher) it is recycled.By the cell (1 × 10 of recycling⁶HepG2、5×10⁶HT29 and 1 × 10⁷STC-1 it) hangs Float in the HBSS that 10 μ L include matrigel (1:1).After to cell sampling, by via with O₂/N₂Gas (the ratio of 3:7 Example) the mask of mixture make mouse holonarcosis to apply 2% (v/v) isoflurane, and liver is exposed to surgical operation. The cell mixed with matrigel is slowly injected into liver simultaneously close incisions.After 4 weeks to 6 weeks, confirmed using MR image Tumor size.

1-5, MRI imaging

The MR image of HCC tumour, SNC tumour and CAC tumour is obtained by following method.It is applied and is mixed by mask There is O₂/N₂5% (v/v) isoflurane of gas (ratio of 3:7) makes Animal Anesthesia, and passes through 1.5% to 2% isoflurane of application To keep.

Body temperature is maintained 36 ± 1 DEG C using circulating water heating pad, and continues to monitor breathing during entire sweep time Rate.After obtaining pre- radiography MRI image before radiography enhancing, via tail vein using intravenous injection come transport of H MS (every kg Weight 3mg Mn).After radiography enhancing 3 minutes, 6 minutes, 9 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, it is 6 small When, 12 hours, 24 hours and the MR image after radiography is collected at 48 hours.

In addition, obtaining the MR image of brain and various organs from the mouse with HCC tumour.Obtaining pre- radiography MR image Later, it executes through tail vein intravenous injection HMS (every kg weight 3mg Mn) to obtain MR image.

All in vivo MR imagings are executed using 7T/20MR system (Bruker-Biospin, Fallanden, Switzerland), 7T/20MR system is with can reach the 20cm tilting gearing of 400mT/m in 100 μ s rise time.Firstly, passing through orthogonal voice coil (35mm id) and fast spin echo (FSE) T1 weighted mri sequence ((repetition time TR)/echo time (TE)=380/ 7.7ms tests serial number NEX=3, echo train length=2, plane 200 × 200mm of intrinsic resolution, thickness 1mm and 14 layers) it will The MR image of mouse liver is received for exciting with signal.After HMS is injected 30 minutes, FSE T1 weighted mri sequence is used (TR/TE=380/7.7ms, NEX=8, echo train length=2, plane 100 × 100mm of intrinsic resolution, thickness 1mm and 14 Layer) to measure MR image again.

In order to obtain brain MR image, using birdcage coil (72mm i.d.) for exciting, and the decoupling of activation is used Phased array coil is received for signal.To FSE T1 weighted mri sequence (TR/TE=325/7.7ms, NEX=12, echo train Length=2, plane intrinsic resolution 100 × 100mm, 12 layers) execute brain imaging.

1-6, immunostaining (Anti-prohibin antibody)

Hepatic tissue is extracted from animal model for cancer and leaching immediately is solid in 10% formalin solution.The group for soaking solid is woven For at wax stone, it is cut into 4 μ m thicks and is fixed on glass slide.The tissue being fixed on glass slide is dewaxed, is then usedantiprohibitin(abcam, mitochondrial markers) and hematoxylin (nuclear targeting) dye it.In dyeing Afterwards, the tissue of dyeing is mounted on anti-dye mounting medium and uses Olympus DP70 digital phosphor microphotographic camera (Olympus, Melville, NY) makes its visualization.

1-7, NADH- tetrazole restore enzyme dyeing

Hepatic tissue is extracted from animal model for tumour, and is embedded to OCT embedding medium (Optimal Cutting immediately Temperature compound, OCT mixture, Tissue Tek, Sakura, France) in be placed in cooling different of liquid nitrogen Until cutting in pentane.Frozen samples are cut into using freezing-microtome (Thermo Scientific, Kalamazoo, MI) 5 μm of size, and 0.4mg/mL NADH and 0.8mg/mL NBT (4- nitro indigo plant tetrazolium chloride) is added dropwise, then at 37 DEG C Reaction 30 minutes to 60 minutes.After washing, sample is mounted on anti-dye mounting medium and by identical in a manner of example 1-6 Mode make its visualization.

Example 2, the Mn relaxation properties of hollow manganous silicate (HMS) nano particle and T1 radiography are enhanced propertied

The Mn relaxation properties and T1 radiography of the HMS prepared in test sample 1-2 according to example 1-3 are enhanced propertied.It uses TEM come observe synthesis nano particle and from the buffer solution of pH 5.0 separate nano particle (Fig. 1 a and Fig. 1 b).

As Fig. 1 c and Fig. 1 d's as a result, when the Mn comprising 19.3wt% PEG modify HMS be dipped in pH 5.0 buffering it is molten When in liquid, releasable all Mn²⁺It releases immediately, and increases to free Mn in 15 minutes²⁺The maximum concentration of ion. On the contrary, when the HMS of PEG modification is dipped in the buffer solution or distilled water of pH 6.0, Mn²⁺Ion very slowly discharges (Fig. 1 c).In addition, the t1 weighted image in distilled water is not influenced by HMS.However, when HMS is added to the buffer solution of pH 5.0 When, T1 signal strength increases quickly, and therefore, t1 weighted image is brightly displayed after 15 minutes of HMS are added.

These results indicate that the Mn flowed out from HMS²⁺Contrast enhancing effects are significant in an acidic solution.

The evaluation of the radiography enhancing pattern of HMS in example 3, HCC

Firstly, HMS is injected into (3mg/kg weight) human hepatocellular carcinoma (HCC) model, and after injecting HMS It is observed in 0.5 hour to 24 hours, a possibility that evaluate HMS as contrast agent for distinguishing liver tumour.

As Fig. 2's as a result, observing apparent radiography enhancing in 0.5 hour to 6 hours after injecting HMS, then Slowly start to brighten from neighboring area to central area in 6 hours to 24 hours, and necrotic zone is not enhanced.

It can be construed to, these results indicate that (primary cell (the Cooper in library during initial 0.5 hour to 6 hours Cell the phase) is absorbed), Mn²⁺Ion is from the nano particle release absorbed in Kupffer cell, then in 6 hours to 24 hours (gallbladders Juice acquisition time) in be discharged into Mn in hepatic tissue²⁺Ion is absorbed and be discharged into bile by liver cell, that is, ion is by liver cell Absorption is then released into bile.

Example 4 passes through the characterization (root of the tissue property of liver tumour after HMS progress radiography enhancing in t1 weighted image According to the differentiable diagnosis effect of vascular distribution, cell density, mitochondria activity and liver cell affinity)

Based in example 3 as a result, three kinds of liver tumour models (the primary HCC, metastatic prepared in test sample 1-4 SNC and CAC) t1 weighted image radiography enhancing pattern difference.

Such as Fig. 3 a's as a result, during injecting 24 hours after HMS, HCC model show the signal strength of tumour from Initial stage until gradually increase for 24 hours.Small intestine neuroendocrine carcinoma (SNC) model shows the signal strength of tumour from opening Begin still to keep by force by 24 hours.CAC (colonic adenocarcinoma, adenocarcinoma of colon) shows the signal strength of tumour From starting by 24 hours to keep weakly.Fig. 3 b shows after injecting HMS the similar pattern until 24 hours.More specifically, HCC Model shows that after injecting HMS, interimage enhancing in 1 hour becomes heterogeneous from tumour periphery, and gradually with uniform enhancing The central area of tumour is filled into until 24 hours.In SNC model, during the radiography of the peripheral portion of tumour enhancing degree is higher than The radiography of center portion point enhances degree, but the enhancing region for enhancing region ratio HCC model is big.It is compared with HCC model, although SNC The radiography enhancing of tumour in model slightly increases compared with HCC model, but the enhancing of the radiography of the tumour in SNC model is by force It keeps to 24 hours.Finally, observing CAC model in 1 hour to 24 hours after HMS injection.With HCC model or SNC model It compares, the radiography enhancing degree of the tumour in CAC model remains significant decrease state, and shows in the neighboring area of tumour The enhancing of thin ribbon shaped radiography is shown.As a result, the radiography enhancing degree of tumour reduces at 24 hours later in all three liver cancer (as shown in the image after 48 hours and 5 days), and the enhancing of hepatic parenchymal radiography is most strong after 1 hour to 3 hours Ground increases.

As a result, currently leading Gd to be used³⁺Class MRI contrast agent is for simple in the short time after injection of contrast agent Observe the contrast agent of the dynamic studies of blood distribution in ground.On the other hand, the experiment goes out as the result is shown, is used in the present invention The contrast agent of HMS shows after 24 hours in being injected into liver tumour and is observed very according to the distribution of manganese ion in tumour Different contrast enhanced images.

That is, because HMS has according to tissue characteristics and blood distribution and acquired character radiography enhancing pattern High likelihood, it is expected that HMS have the effect of to the antidiastole of liver tumour it is excellent.In this respect, if HMS enhancing MRI, which is imaged in each tumour, provides property feature, then HMS can be referred to as disease specific MRI contrast agent.

Tissue characteristics (vascular distribution, cell density, the line grain for the liver tumour that example 5, basis are dyed by using mitochondria Body activity and liver cell affinity) differentiable diagnosis effect

Manganese ion is deposited in mitochondria present in all cells.Therefore, if line grain in certain organs or disease Body activity is high, then is transferred to organ or disease from the manganese ion that HMS discharges and is largely deposited, thus the table in t1 weighted image Reveal high signal intensity.Based on exemplary as a result, in order to which whether the MRI that the radiography enhancing tested through HMS obtains reflects disease The tissue specificity of disease, measures mitochondria activity and shows in Fig. 4.

Firstly, HCC model shows the dyeing that the dye levels in tumour are higher than in liver parenchyma according to the distribution of mitochondria Degree, and CAC model shows the dye levels that the dye levels in liver parenchyma are higher than in tumour.In this case, it dyes The higher region of degree includes more mitochondrias.There are more mitochondrias in some tissues, this indicates the cell of tissue The quantity of Mitochondria it is higher or tissue in cell density it is higher.

In addition, such as the knot for dyeing (nuclear targeting) and antibody staining analysis vascular distribution and cell density by hematoxylin Fruit, in HCC model, HCC cell density is higher than hepatic parenchymal cell density.In CAC model, in the central area of CAC There are a large amount of meronecrosises, and the cell density of around the area is lower than hepatic parenchymal cell density.

In HCC model, than there are more mitochondrias in liver parenchyma in HCC, and t1 weighted image compares in HCC Higher signal strength is shown in liver parenchyma.On the contrary, in CAC model, it is more multi-thread than existing in CAC in liver parenchyma Plastochondria, and t1 weighted image in CAC than showing lower signal strength in liver parenchyma.In CAC model, due to swollen Oncocyte is close or surrounding normal cell is in compressive state and causes the cell density in the region high, thus it is speculated that goes out band-like radiography Enhancing.

Finally, in SNC model, although the dye levels in liver parenchyma and tumour are not high and are not significantly different, It is compared with liver parenchyma, tumour shows very high radiography enhancing degree.This, which can deduce SNC model, is contaminated in mitochondria With the vascular tumor of very high vascular distribution in color.

In addition, HCC model shows the radiography enhancing increased over time, but SNC model keeps relative constant height to make Shadow enhancing.In HCC model, because manganese ion is absorbed into cell but is discharged into gallbladder due to the maintenance of liver cell affinity Road (Ni, Y, et al., Tumor models and specific contrast agents for small animal Imaging in oncology, Methods 48,1930-1938 (2015)), so the concentration of manganese ion is gradually increased.In In SNC model, because manganese ion is only absorbed into cell, and due to not having liver cell affinity without being discharged into bile duct In, so a large amount of manganese ion is gathered from the initial stage.

Therefore, the liver tumour of three types has various tissue characteristics, and such as vascular distribution, cell density, mitochondria are living Property and each liver tumour liver cell affinity, make to be provided in t1 weighted image in specific imaging time with characteristic The liver tumour of the three types of shadow image.

Example 6 is distributed by using the liver cancer specific blood vessels of FSE t1 weighted image

Based on the above results, FAST SPIN echo (FSE) T1 that 3 minutes to 15 minutes obtain after injecting HMS is weighted Image shows the contrast enhancing effects to the liver cancer of three types.

As Fig. 5's as a result, these are the result shows that the HMS main quilt (3 minutes to 15 minutes after injecting HMS) at this stage Kupffer cell absorbs, therefore occurs rapider in the radiography Enhanced time radiography of liver tumour enhancing pattern, wherein liver tumour Radiography enhance by discharging manganese ion.Therefore, the radiography as caused by vascular distribution enhances ratio by initial stage line grain Radiography enhancing reflection caused by body activity is much.

More specifically, compared with the image obtained after injecting 1 hour after HMS, HCC model and SNC mould at this stage The radiography enhancing pattern of tumour in type is more distributed in neighboring area.However, only being observed band-like in CAC model Weak radiography enhancing, and tumour itself do not show radiography enhance.

Result as the above results, it is contemplated that vascular distribution can contribute to the enhancing pattern of three types liver tumour, this It can be used for the antidiastole of liver tumour.

Example 7, by the necrosis of HCC after NADH- tetrazolium () reductase staining evaluation hepatic artery ligation, (cell is dead Die) degree

Among the method for the treatment of liver cancer, TACE is controlled by blocking blood to flow to tumour and blocking the arteria hepatica of patient The method for treating HCC necrosis.In order to confirm neoplasm necrosis (cell death) and t1 weighted image after HCC model mice hepatic artery ligation It whether there is correlation between HMS signal strength at any time, measure work using NADH- tetrazolium () reductase colouring method NADH- dehydrogenase activity (Berardi et al., the Neurol Res.2008 of intracellular mitochondrial in cell；30(2):160- 9)。

More specifically, the stomach wall of dissection HCC model mice, and ligature left hepatic artery and be closed stomach wall.At 2 hours and 24 HMS is applied to animal after hour.After injection HMS 2 hours and 24 hours, MRI image is obtained.

As shown in Figure 6 as a result, the t1 weighted image that injection in 2 hours obtains after HMS 1 hour after ligation confirms Tumour shows that radiography enhances on periphery.Because the degree of tumour cell bulk dyeing is weaker than control group, it is found that survival Tumour cell.Therefore, by combining coloration result and MRI as a result, because blood flow passes through vena portae hepatica confession in hepatic artery ligation Tumour should be arrived, and the tumour cell only survived in the region for being supplied with blood is dyed using NADH dyeing, therefore in T1 Identical region shows that radiography enhances in weighted image.

In addition, in the t1 weighted image that injection in 1 hour and 4 hours obtains after HMS 1 hour after ligation, in tumour Do not observe that radiography enhances completely, and there is no the cells with NADH dyeing dyeing, it was confirmed that complete tumor necrosis.

As the result for summarizing the result, it is contemplated that the mitochondria activity of HMS can be used for evaluating the cancer cellular necrosis of HCC (cell death), thus by evaluation anticancer therapy after meronecrosis and TACE be used to determine therapeutic effect.

Foregoing description of the invention is illustrative.It will be appreciated by those skilled in the art that of the invention not changing In the case where spirit or essential attributes, the present invention can be easily modified in other specific forms.

Example 8, the [email protected] as MRI contrast agent₂The preparation of NP

8-1、[email protected]₂(MnO-SiO₂- 12hr) preparation and Mn²⁺The measurement of release time

(1)[email protected]₂(MnO-SiO₂- 12hr) nano particle preparation

MnO NP is used as Mn²⁺Ion source.These particles are not dissolved at condition of neutral pH (~pH7), and in low ph condition It is dissolved under (pH 4~5).

Firstly, in order to reduce Mn²⁺It from the rate of release of particle, synthesizes MnO NP (15nm), and uses reverse micelle lotion mould Plate makes the MnO NP (15nm) of synthesis be covered with silica shell (Fig. 7).

In order to synthesize 15nm MnO nano particle (NP), the Mn- oleic acid complex compound of 1.24g is dispersed in the 1- 18 of 10g In carbene solvent, and under conditions of stopping oxygen and moisture, solution to be heated to together with stirring under 5 DEG C/min of rate 300℃.Then, solution is heated 1 hour at 300 DEG C, is cooled to room temperature, and be centrifuged to separate particle.With hexane and The particle of acetone washing separation, and the MnO nano particle of the synthesis having a size of 15nm is dispersed in hexamethylene and is stored.

Then, MnO nano particle is surrounded with silica shell to obtain [email protected] by using reverse micelle emulsion template₂ Nano particle.Specifically, after in the hexamethylene that the MnO nano particle of 20mg is dispersed in 40mL, the surface of 3.2mL is added Activating agent IGEPAL CO-520, and the mixture is stirred 30 minutes.Hereafter, sequentially add 0.3mL's with 30 minutes intervals NH₄The TEOS (tetraethyl orthosilicate) of OH solution and 0.8mL.By solution stir one day after, the methanol of 5mL is added with separate water layer and Organic layer.Water layer is separated and is centrifuged to separate particle, with ethyl alcohol and water washing particle to obtain by two with a thickness of 10nm The [email protected] that silica shell surrounds₂Particle.

(2)Mn²⁺The measurement of release time

Silica shell prevents Mn²⁺It is released immediately in low ph conditions and improves point of particle under conditions of neutral ph It dissipates.

At room temperature using the 16mg's in citric acid (salt) buffer or distilled water of the pH 5 or 6 for being dispersed in 32mL [email protected]₂Suspension quantify Mn²⁺Ion is from [email protected]₂Rate of release.0 minute, 15 minutes, 1 Every kind of sample of 4mL is collected in hour, 2 hours, 4 hours, 6 hours, 8 hours and 12 hours, and is centrifuged 10 points with 15,000rpm Clock, to obtain nano particle (NP) from suspension.Then, supernatant is filtered with the injecting type filter in 0.2 μm of (micron) aperture Liquid.Mn in the supernatant of measurement filtering is analyzed by ICP-AES²⁺The concentration of ion, and use Mn in supernatant²⁺The matter of ion The quality relative to manganese in sample is measured to calculate Mn²⁺The rate of release of ion.

As shown in figure 8b, have citric acid (salt) of the nano particle of the silica shell with a thickness of 10nm in pH 5 slow Slow release Mn in fliud flushing²⁺, and spend 12 hours Mn from MnO NP release 80wt%²⁺Ion.On the other hand, in pH 6.0 Citric acid (salt) buffer or pH 7 distilled water in only discharge the Mn of 10wt%²⁺Ion.Then, have with a thickness of 10nm's The [email protected] of silica shell₂NP is expressed as [email protected]₂Or MnO-SiO₂- 12hr (Fig. 8 b).

8-2、[email protected]₂(MnO-SiO₂- 2hr) preparation and Mn²⁺The measurement of release time

(1)[email protected]₂(MnO-SiO₂- 2hr) nano particle manufacture

In order to manufacture under the conditions of lower pH than [email protected]₂Quickly discharge Mn²⁺The particle of ion adjusts dioxy The porosity and thickness of SiClx shell.

In order to synthesize the nano particle with the silica shell with a thickness of 8nm, the 0.8mL TEOS in example 8-1 is replaced It is changed to 0.72mL TEOS and 0.08mL C18TMS (n-octadecane base trimethoxy silane).

Then, in order to increase the porosity of silica shell, organosilan (C18TMS, n-octadecane base trimethoxy are used Base silane) it is used as pore creating material.The silane of pore creating material (90vol%TEOS+10vol%C18TMS) is mixed in silica shell More holes are generated, so that solution be allowed to penetrate into core MnO.

(2)Mn²⁺The measurement of release time

It is acid molten that the porosity of the increase of silica shell or reduced thickness are easier exposed to MnO nano particle Liquid, to increase Mn²⁺Rate of release.

As shown in fig. 8 c, the [email protected] of the silane encapsulating mixed with pore creating material₂Citric acid (salt) of the NP in pH 5.0 is slow It rushes in solution and discharges the Mn of 80wt% in 2 hours²⁺.Then particle is named as [email protected]₂Or Mn-SiO₂-2hr。

[email protected]₂NP and [email protected]₂NP has the silica shell of comparable thickness, but due to difference Porosity, show very different Mn in citric acid (salt) buffer of pH 5.0²⁺Rate of release (Fig. 8 c).

8-3、[email protected]₂(MnO-SiO₂- 0.5hr) manufacture

(1)[email protected]₂(MnO-SiO₂- 0.5hr) nano particle manufacture

In order to reduce the thickness of silica shell in the manufacturing method of example 8-1, with SiO₂The technique for coating MnO core Such as [email protected] is only added in period₂Silica precursor half amount (0.4mL) silica precursor (TEOS).The silica shell thickness of nano particle is 5nm.

(2)Mn²⁺The measurement of release time

As shown in Fig. 8 d, [email protected]₂The Mn of 80wt% is discharged in 30 minutes at pH5.0²⁺.Hereafter, particle It is named as [email protected]₂(Mn-SiO₂-0.5hr)。

[email protected] in example 8-2₂With [email protected]₂PH 6.0 citric acid (salt) buffer soln or The Mn of 10wt% is only discharged in distilled water²⁺(Fig. 8 c and Fig. 8 d).For the nano particle of all designs, with methoxy group PEG is for effectively dispersion under aqueous conditions.

The diversity ratio of the rate of release and characteristics of nanoparticles under the conditions of similar pH of 8-4, liver Kupffer cell Compared with

As example 8-1 is measured into example 8-3, each particle is directed under citric acid (salt) buffer conditions of pH 5 It collects and compares Mn²⁺The rate of release of ion.As a result it shows in Fig. 8 e.

When nano particle is injected into (internal condition) in vivo, they are absorbed by the Kupffer cell in hepatic tissue. Know that the pH value in Kupffer cell is about pH 5.In other words, in this experiment, by comparing the conditions in vitro in 5 buffer of pH Under every kind of particle Mn²⁺The rate of release of ion, prediction every kind of particle when particle is absorbed into Kupffer cell (internal) Mn²⁺The rate of release of ion, the conditions in vitro of 5 buffer of pH and the pH condition of Kupffer cell are similar.

8-5, release Mn²⁺Contrasting effects of the ion in T1 weighted mri

In order to determine the Mn discharged from nano particle²⁺What kind of contrasting effects ion pair T1 weighted mri shows, and obtains simultaneously The Mn for citric acid (salt) buffer (pH 5.0) in test sample 8-1 obtained in specific time is shown in fig. 9 a²⁺ The T1 weighted mri of the supernatant soln of the rate of release of ion.

According to Fig. 9 a, MnO-SiO₂The Mn of -0.5h particle²⁺Ion largely is discharged in 0.5 hour, therefore 0.5h extremely 12h shows almost the same contrast intensity.Because of MnO-SiO₂The Mn of -12h particle²⁺The rate of release of ion is relatively low, institute It is gradually increased as time goes by with the contrast intensity of MRI.

The reduction of contrast signal intensity of the image of shooting is digitized (R₁, unit=second (s)), and the curve in Fig. 9 b is shown In figure.Based on Fig. 9 b almost Mn with every kind of particle shown in Fig. 8 e²⁺The curve of the rate of release of ion is consistent, infers release Mn²⁺Ion can increase the contrasting effects of t1 weighted image.

Then, Mn²⁺The concentration of ion and the contrast intensity R of image₁Between relationship show in the curve of Fig. 9 c.The song The slope of line is expressed as r₁, and be unique value for every kind of material.Confirmation is that three kinds of particles have range 7.5~8.2 Interior similar slope, and by with r₁The pure Mn that value is about 7.4²⁺Ion (MnCl₂Aqueous solution) it compares, reconfirm radiography Effect is because of Mn²⁺Ion and increase.

Example 9, internal MRI data

The manufacture of 9-1, HCC animal model

In order to test MRI how design [email protected]₂The different Mn of contrast agent²⁺How to change under rate of release, invents People is by human hepatocellular carcinoma (HCC) insertion mouse to manufacture vivo tumor model.Specifically, the manufacturing method of HCC animal model with Mode in example 1-4 is identical.

9-2, MRI imaging

Hereafter, in addition to using [email protected]₂(Mn-SiO₂- 0.5hr) nano particle and after injecting nano particle Except 15 minutes, 30 minutes, 45 minutes, 1 hour, 4 hours, 8 hours and 24 hours collection MRI images, with the phase of example 1-5 The MRI image of HCC is obtained with method.

9-3, [email protected] is injected in HCC model₂MRI image variation later

According to the method for example 9-2, [email protected] is injected in HCC model₂(MnO-SiO₂- 0.5h) obtain later MRI image is shown in figure loa.

(1) the radiography variation of normal liver tissue

As illustrated in fig. 10 a, when by [email protected]₂When being injected into HCC model (liver parenchyma), a series of T1 weightings The enhancing of MRI radiography started at 30 minutes and is maximized after 1 hour.After 1 hour, hepatic parenchymal radiography enhancing gradually subtracts It is few, and it is completely recovered to preform injection level after 8 hours.

That is, reaching the [email protected] of liver by blood vessel₂NP by endocytosis into Kupffer cell, and it is big Part Mn²⁺Ion discharges in 30 minutes, therefore starts radiography enhancing occur.After 1 hour, Mn²⁺Ion is no longer general from library Not cell discharges, and because the Mn that liver cell absorbs²⁺Ion is discharged into cholangiole by liver and gallbladder approach, so radiography enhances It reduces.

(2) the radiography variation of HCC tumor tissues

HCC tumor tissues brighten since neighboring area after 1 hour, and HCC tissue is gradually shifted in radiography enhancing Central area (Figure 10).After injection 24 hours, necrosis part remains unchanged and keeps dark radiography, and entire HCC The radiography of tissue increases.

HCC tumor tissues lack Kupffer cell and not direct endocytosis nano particle.However, the liver cell of HCC tissue With mitochondria activity, therefore the Mn discharged from the Kupffer cell around normal tissue can be absorbed in they²⁺.Therefore, HCC is swollen The radiography enhancing of tumor tissue starts from the outer region of tumour, and when the Kupffer cell in normal tissue after about 1 hour Discharge Mn²⁺Gradually brighten when reaching maximum horizontal, and with from around liver normal tissue parenchymal tissue's regional diffusion and The Mn of discharge²⁺Into tumor tissues, blast to central area.

(3) in [email protected]₂The standard of liver normal tissue and HCC tumor tissues is distinguished in the case where contrast agent

It therefore, can be according in intravenous injection [email protected]₂The radiography of the MRI image obtained at any time later enhances The brightness change pattern of image distinguishes normal liver tissue and HCC tumor tissues.

Normal tissue shows high radiography enhancing from injection in 30 minutes to 1 hour, and in 1 hour to 24 hours Show low radiography enhancing.HCC tumor tissues are dark (black) or only on periphery in 30 minutes to 4 hours from injection time Region brightens, and high radiography enhancing is shown in 4 hours to 24 hours.

9-4, [email protected] is injected in HCC model₂MRI variation afterwards

According to the method for example 9-2, [email protected] is injected in HCC model₂The MRI image obtained later is shown In Figure 10 b.

(1) hepatic parenchymal radiography variation

By by [email protected]₂Intravenously it is administered to the MRI image of HCC model acquisition and from example 9-3 [email protected]₂The image (Figure 10 a) of acquisition is not significantly different.

In normal liver parenchyma, radiography enhancing from intravenous injection [email protected]₂It rises in 30 minutes and starts to increase, and And reach maximum horizontal after four hours.However, with [email protected]₂Difference still has some radiographies after 8 hours Enhancing.

With [email protected]₂Equally, [email protected]₂Also it is gradually increased the radiography of normal tissue.This is because passing through The Mn of the nano particle of normal tissue endocytosis²⁺Rate of release is higher than the Mn of normal tissue²⁺Rate of release.On the other hand, Mn²⁺In It is slowly accumulated in liver cell, and than [email protected]₂It is kept as much late.Therefore, maximum radiography enhancing slowly occurs and delays It is reduced to preform injection horizontal slowly.

(2) the radiography variation of HCC tumor tissues

In HCC tumor tissues, radiography just increased from injecting nano particle until 1 hour, but after four hours from swollen The outer region of tumor tissue starts to increase and gradually brighten to inside tumor until 24 hours.

The time needed for radiography enhancing is by the Mn from MnO NP in the outer region of HCC tumor tissues²⁺Ion is released It puts rate to determine, the Kupffer cell endocytosis that the MnO NP is located in normal tissue.Due to [email protected]₂With than [email protected]₂Relatively lower rate of release discharges Mn²⁺Ion, therefore the radiography of HCC tumor tissues neighboring area enhances table Reveal than [email protected]₂Slowly.

(3) in [email protected]₂The standard of liver normal tissue and HCC tumor tissues is distinguished in the case where contrast agent

Therefore, according to intravenous injection [email protected]₂Enhance image at any time in 45 minutes to 24 hours interimages later Between brightness change pattern, normal liver and HCC tumor tissues can be distinguished.

Normal tissue shows high radiography enhancing in 45 minutes to 4 hours, and shows in 4 hours to 24 hours The enhancing gradually decreased.HCC tumor tissues are presented black in 45 minutes to 4 hours, but interimage enhancing in 4 to 24 hours from Outside is gradually diffused into inside tumor region with high intensity.

9-5, [email protected] is injected in HCC model₂MRI changes later

According to the method for example 9-2, [email protected] is injected in HCC model₂The MRI image obtained later is shown In Figure 10 c.

(1) hepatic parenchymal radiography variation

As shown in fig. 10 c, with first two [email protected]₂Contrast agent and [email protected]₂Contrast agent is different, passes through [email protected]₂A series of T1 Contrast-enhanced MRIs between the liver parenchyma of the HCC model of control, which started to generate after 45 minutes, to be made Shadow enhancing, and brightness is maintained at phase same level, until 8 hours without significant changes (Figure 10 c).At 24 hours later, [email protected]₂The radiography enhancing of mediation does not return to preform injection level.

(2) the radiography variation of HCC tumor tissues

Whole HCC tissue was in black up to 4 hours, and occurred the radiography enhancing of thin peripheral shape after 8 hours.So And even if at 24 hours later, the radiography enhancing of the central part of HCC tissue is not also extended.

Due to compared with first two contrast agent, Mn²⁺Rate of release is lower under acidic environment, therefore [email protected]₂ Even if also showing that slower radiography enhances by endocytosis in Kupffer cell.Since HCC is to Mn²⁺The absorption rate of ion and Mn²⁺The rate that ion is discharged from Kupffer cell is in equilibrium state, therefore in the normal tissue by [email protected]₂It mediates Radiography enhancing remain [email protected]₂Radiography enhancing shown in neighboring area it is most slow in three kinds of NP.

(3) in [email protected]₂The standard of liver normal tissue and HCC tumor tissues is distinguished in the case where contrast agent

Therefore, according in intravenous injection [email protected]₂Later 45 minutes to 24 hours interimages enhancing image with The pattern of the brightness change of time can distinguish normal liver tissue and HCC tumor tissues.

Normal tissue shows high radiography enhancing in 45 minutes to 8 hours, and shows in 8 hours to 24 hours The radiography enhancing gradually decreased.Dark (black) is presented in 45 minutes to 8 hours or only has in neighboring area for HCC tumor tissues Radiography enhancing, and high radiography enhancing is presented in entire tumor region in 8 to 24 hours.

Example 10, the confirmation of long-term radiography enhancing later in injecting contrast medium into HCC model

According to the identical method of example 1-5, the [email protected] that will be synthesized in aforementioned exemplary₂Contrast agent, [email protected] pSiO₂Contrast agent and [email protected]₂Each of contrast agent is administered to primary liver tumor model (HCC model), then right The MRI image measuring signal (paravision software version 5.0, Bruker-Biospin) of each organ.Measurement length at any time As a result phase contrast enhancing effects and discharge are shown in Figure 11 a to Figure 11 e.

In normal liver, from application [email protected]₂(Mn-SiO₂- 0.5hr) rise 30 minutes after radiography it is obviously significant And at 45 minutes, signal was most strong, and gradually decreased after 1 hour.From injection [email protected]₂(Mn-SiO₂-2hr) After rising 45 minutes, contrasting effects are kept, and peak signal was kept from 1 hour to 4 hours, was then reduced.From injection [email protected] 10nm-SiO₂(Mn-SiO₂- 12hr) rise 45 minutes after, there are contrasting effects, and contrasting effects maintained until 8 hours and Reduce (Figure 11 a) at 24 hours later.

In tumor region, from application [email protected]₂(Mn-SiO₂- 0.5hr) from after 1 hour, normal region Contrasting effects reduce, the contrasting effects of tumor region increase and keep to 24 hours after 1 hour.From application [email protected] pSiO₂(Mn-SiO₂- 2hr) from after 4 hours, the contrasting effects of normal tissue reduce, then the radiography of tumor tissues Effect increases (Figure 11 b).From application [email protected]₂(Mn-SiO₂- 12hr) from after 8 hours, normal region is made Shadow effect reduces, and then the contrasting effects of tumor region are kept to 24 hours (Figure 11 b).

In the case where kidney, from administration of contrast agents after 4 hours, the contrasting effects in kidney increase after four hours Add, this indicates from administration of contrast agents Mn after 4 hours²⁺Ion is discharged by kidney approach.(Figure 11 c).

Specific contrasting effects (Figure 11 d) are not observed in spleen.

In intestines, contrasting effects are in application [email protected]₂(Mn-SiO₂- 0.5hr) increased later to 45 minutes, then It reduces.In [email protected]₂(Mn-SiO₂- 2hr) in the case where, contrasting effects increase after 4 hours from application, so After reduce.In [email protected]₂(Mn-SiO₂- 12hr) in the case where, contrasting effects increased to 1 hour after administration, so Reduce (Figure 11 e) afterwards.

Example 11, selection nano particle are to control Mn²⁺Rate of release and the radiography for optimizing liver tumour type

In order to control Mn²⁺Rate of release, the present inventor use the three kinds of MnO prepared in example 8₂@SiO₂NP has carried out reality It tests, with the angiogenesis of the pathologic characteristic of the various tumor models of determination (HCC, SNC, CAC) and blood vessel.For this purpose, obtaining body Interior MRI result.

The manufacture of 11-1, animal model

In order to manufacture the tumor model of allograft mouse, small intestine neuroendocrine carcinoma (SNC) is used in an experiment With adenocarcinoma of colon (CAC) and hepatocellular carcinoma (HCC) as the representative of the cell that mitochondria is insufficient and mitochondria lacks Non- liver cell metastatic tumo(u)r model.

Specifically, SNC (STC-1) animal model and CAC (HT29) animal mould are prepared in a manner of identical in example 1-4 Type.

11-2, the MRI variation in CAC (HT29) model after injection nano particle

According to the method for example 9-2, by [email protected]₂、[email protected]₂With [email protected]₂It injects respectively To CAC model, 24 hours MRI images are obtained.MRI image is shown in Figure 12.

(1)[email protected]₂Injection

It is shown in Figure 12 a in injection [email protected]₂The image obtained later.According to Figure 12 a, normal tissue is being infused Show that radiography enhances from when penetrating in 15 minutes to 45 minutes, radiography enhancing hour is gradually decreased from 1 hour to 24, and is restored to Inject the level before contrast agent.

On the other hand, it in tumor tissues, only observes and makes at the fringe region of tumour in 15 minutes to 24 hours Shadow enhancing.

It is changed with time pattern by analyzing MRI image, contrast agent can be used for distinguishing normal tissue and CAC tumor group It knits.Normal tissue shows high radiography in 15 minutes to 45 minutes, and hour gradually decreases from 1 hour to 24.Tumor tissues Black is kept in 15 minutes to 24 hours or only keeps bright in neighboring area.

(2)[email protected]₂Injection

It is shown in Figure 12 b in injection [email protected]₂The MRI image obtained later.According to Figure 12 b, normal tissue Show that radiography enhances in 30 minutes to 4 hours, and after four hours, radiography enhancing gradually decreased and at 24 hours The level being restored to before injection contrast agent later.

On the other hand, the radiography of tumor tissues, which enhances, increases since 45 minutes and increases since borderline tumor, and Two layers of inside tumor is formed, wherein internal layer is that black enhances without radiography, is had directly about the outer layer of black portions Some radiography enhancings inwardly spread as HCC.

It is changed with time pattern by analyzing MRI image, contrast agent can be used for distinguishing normal tissue and CAC tumor group It knits.Normal tissue shows the enhancing of high radiography in 30 minutes to 4 hours, and show after 4 hours to 24 hours by The radiography enhancing gradually reduced.Tumor tissues keep black in 30 minutes to 24 hours or only keep bright in neighboring area It is bright.

(3)[email protected]₂Injection

It is shown in Figure 12 c in injection [email protected]₂The MIR image obtained later.According to Figure 12 c, normal tissue Show that radiography enhances in 15 minutes to 4 hours, and radiography enhancing gradually decreases in 8 hours to 24 hours, and extensive The level before contrast agent injection is arrived again.

On the other hand, in tumor tissues, hour only continuous edge enhancing of tumour from 1 hour to 24.

It is changed with time pattern by analyzing MRI image, contrast agent can be used for distinguishing normal tissue and CAC tumor group It knits.Normal tissue shows the enhancing of high radiography in 15 minutes to 4 hours, and hour shows from 8 hours to 24 and gradually drop Low radiography enhancing.Black is presented in 15 minutes to 4 hours or only keeps bright in neighboring area for tumor tissues, and Only observe that radiography enhances in tumor boundaries in 8 hours to 24 hours.

11-3, the MRI image variation in SNC (STC-1) model after injection nano particle

According to the method for example 9-2, in [email protected]₂、[email protected]₂With [email protected]₂It injects respectively After into SNC model, for the MRI image of acquisition in 24 hours at any time.MRI image is shown in Figure 13.

(1)[email protected]₂Injection

It is shown in Figure 13 a in injection [email protected]₂The MRI image obtained later.According to Figure 13 a, normal tissue exists It shows that radiography enhances in 15 minutes to 4 hours, and is gradually decreased in interimage enhancing in 4 hours to 24 hours, and restore Level to before contrast agent injection.

On the other hand, tumor tissues showed similar with normal tissue in 30 minutes to 1 hour, but at 4 to 24 hours Interimage enhancing continues.

It is changed with time pattern by analyzing MRI image, contrast agent can be used for distinguishing normal tissue and SNC tumor group It knits, two kinds of groups, which were woven in 15 minutes to 1 hour, to be distinguished well, but in 4 hours tumor tissues after 24 hours In radiography enhancing gradually appear and brighten.

(2)[email protected]₂Injection

It is shown in Figure 13 b in injection [email protected]₂The MRI image obtained later.According to Figure 13 b, normal tissue Radiography enhancing and no high intensity are shown in 15 minutes to 1 hour, and do not observe radiography in 4 hours to 24 hours Enhancing.

On the other hand, tumor tissues after injection of contrast agent 15 minutes to 1 hour it is darker to be rendered as compared with low-intensity, but It presents brighter than normal tissue, and is persistently brightened in 4 hours to 24 hours with high intensity.

It is changed with time pattern by analyzing MRI image, contrast agent can be used for distinguishing normal tissue and SNC tumor group It knits, and is difficult to clearly distinguish normal tissue and tumor tissues in 15 minutes to 1 hour.From 4 hours to 24 hour, normally Tissue shows low radiography enhancing, and tumor tissues show high radiography enhancing.

(3)[email protected]₂Injection

Injection [email protected] is shown in Figure 13 c₂The MRI image obtained later.3c referring to Fig.1, normal tissue exist Radiography enhancing is shown in 30 minutes to 1 hour and does not observe that radiography enhances without high intensity, and in 4 to 24 hours.

On the other hand, tumor tissues show low radiography enhancing in 15 minutes to 1 hour, but stronger than normal tissue, And hour persistently increase contrast intensity from 4 hours to 24.

It being changed with time pattern by analyzing MRI image, contrast agent can distinguish normal tissue and SNC tumor tissues, And it is not easy to distinguish two kinds of tissues in 15 minutes to 1 hour, but in 4 hours to 24 hours, normal tissue shows low Radiography enhancing, and tumor tissues show high radiography enhancing.

11-4, for screening HCC, CAC and SNC tumour optimal nano particle test

The radiography of SNC model and CAC model, which enhances pattern, only enhances figure with the radiography of HCC model in normal tissue regions Case is similar.Radiography enhances in [email protected]₂When nano particle is absorbed by the Kupffer cell in normal tissue, and and Mn²⁺'s Rate of release is unrelated.

There is HCC excessive blood vessel, high mitochondria activity and characteristic to discharge approach.Because of the high mitochondria activity of HCC, institute With the Mn discharged from Kupffer cell²⁺Intake efficiently take place in around normal cell.In addition, because passing through HCC approach Periphery excretion also occurs in inside HCC, so the radiography enhancing for observing that the radiography of neighboring area enhances than SNC is slow.

As a result, in HCC diagnostic system, [email protected]₂It is most effective optimal MRI contrast agent.As use [email protected] 5nm-SiO₂When, due to the quick enhancing of normal tissue, the MRI of HCC is dimmed between 45 minutes and 1 hour, so as to table Sign.On the other hand, the MRI of normal tissue is resumed at 24 hours later, while the MRI radiography of HCC is enhanced, to diagnose again Liver cancer.

SNC has many blood vessels and high mitochondria activity, but does not have release approach.Because of high mitochondria activity, by library The Mn that kupffer cell provides²⁺It is effectively absorbed by the normal tissue of surrounding, but Mn²⁺It is constantly accumulated due to there is no excretion pathway It is poly-.Compared with HCC, these cause the whole radiography of SNC to enhance faster.The [email protected] of engineering₂Nano particle is thin by Ku Pufu It is intracellular to gulp down, and SNC tumor tissues and normal tissue almost show that radiography enhances simultaneously.Therefore, with fast recovery rate [email protected]₂With [email protected]₂It is most effective contrast agent in SNC tumor model system.

CAC is low vascular and does not have mitochondria release approach.Since mitochondria lacks, during entire measurement Absorb Mn²⁺It is invalid.This only shows the radiography enhancing of the thin layer in neighboring [email protected]₂Indicate normal tissue In maximum radiography enhancing, therefore observe CAC part normal tissue between significant radiography.On the other hand, due to normal Tissue keep the radiography that slightly improves and after NP injection after 1 hour in long-time without significant change, therefore [email protected] SiO₂It can be used for analyzing program at a slow speed.

Because of Mn²⁺Ion is in Mn²⁺Ion is ingested after normal surrounding tissue release, so CAC model system is shown The radiography behavior slower than normal cell.It is horizontal using preform injection is quickly recovered to due to the high mitochondria activity of HCC and SNC Nano particle be conducive to the clear comparison between tumour and normal tissue.On the other hand, in CAC, due to widened radiography Enhancing, uses [email protected]₂It is advantageous.

To sum up, the analysis that the MRI radiography obtained using the MRI contrast agent of design enhances the pattern that changes with time not only may be used To distinguish tumour and normal liver tissue, and it can characterize and distinguish intracorporal tumor type and origin.