阅读说明：本技术 一种用于阿尔茨海默病致病蛋白的纳米探针及其制备 (A kind of nano-probe and its preparation for Alzheimer disease pathogenic protein ) 是由 蔡静 李立 于 2019-09-29 设计创作，主要内容包括：本发明属于纳米材料和生物医学分子影像技术领域,具体涉及一种用于阿尔茨海默病致病蛋白的纳米探针及其制备；本发明所述纳米探针为多模态纳米探针,由超小铁氧体纳米颗粒、聚乙二醇衍生物和吩噻嗪衍生物制得；所述纳米探针的内核为超小铁氧体纳米颗粒,外层为聚乙二醇片段偶联吩噻嗪衍生物；所述的多模态纳米探针可特异性与β-淀粉样蛋白斑块结合,不仅具有独特的近红外荧光标记增强效应和T<Sub>1</Sub>-T<Sub>2</Sub>磁共振影像对比增强效应；同时该探针还具有超小尺寸、良好的生物相容性、无辐射性和无潜在神经毒性等多种优点,该探针在阿尔茨海默病的早期诊断方面具有良好的应用前景。(The invention belongs to nano materials and biomedical molecular image technical field, and in particular to a kind of nano-probe and its preparation for Alzheimer disease pathogenic protein；Nano-probe of the present invention is multi-modal nano-probe, is made by extra small ferrite nanometer particle, polyethyleneglycol derivative and phenothiazine derivative；The kernel of the nano-probe is extra small ferrite nanometer particle, and outer layer is that polyethylene glycol segment is coupled phenothiazine derivative；The multi-modal nano-probe can be specific in conjunction with beta-amyloid protein patch, not only has unique near-infrared fluorescent label enhancement effect and T 1 ‑T 2 Nuclear magnetic resonance image Contrast enhanced effect；The probe also has the advantages that super-small, good biocompatibility, radiationless property and a variety of without potential neurotoxicity etc. simultaneously, which has a good application prospect in terms of the early diagnosis of Alzheimer disease.)

1. a kind of nano-probe for Alzheimer disease pathogenic protein, it is characterised in that: the nano-probe is multi-modal Nano-probe, kernel are extra small ferrite nanometer particle, and outer layer is that polyethylene glycol segment is coupled phenothiazine derivative；The nanometer The hydrodynamics diameter of probe is 1-30nm；The nano-probe includes the following components calculated by weight:

Extra small ferrite nanometer particle 5-20 parts

1-5 parts of polyethyleneglycol derivative

0.1-2 parts of phenothiazine derivative.

2. being used for the nano-probe of Alzheimer disease pathogenic protein as described in claim 1, it is characterised in that: the pheno thiophene Oxazine derivatives structure are as follows:

Wherein, m is the number of double bond, and n is alkyl chain number；R₁For methoxy base class, carboxyl class, amino, sulfydryl class, Malaysia acyl Imines, nitrine class, biotin or N- succinimido molecule；R₂For hydrogen, hydroxyl, methyl, ethyl, halogen, carboxyl, primary amine Base or tertiary amine groups molecule.

3. being used for the nano-probe of Alzheimer disease pathogenic protein as described in claim 1, it is characterised in that: the poly- second Glycol is the substitution of both-end base, and end group is methoxyl group, amino, sulfydryl, carboxyl, dimaleoyl imino or alkynyl.

4. being used for the nano-probe of Alzheimer disease pathogenic protein as described in claim 1, it is characterised in that: the poly- second two 01 derivatives structural formula are as follows:

Wherein, R₁For alkyl, amino, carboxyl, sulfydryl, phosphoric acid salt, hydroxamic acid or catechol molecule；R₂For methoxy Base class, carboxyl class, amino, sulfydryl class, maleimide, nitrine class, biotin class or N- succinimido molecule；n For 1-200.

5. being used for the nano-probe of Alzheimer disease pathogenic protein as described in claim 1, it is characterised in that: the extra small iron The partial size of ferrite nano particle be 1-10nm, the extra small ferrite nanometer particle the preparation method comprises the following steps: first by presoma with Finish is uniformly mixed, and then mixture is dissolved in solvent and being stirred evenly, the mixture for being mixed with solvent is then heated to 265 DEG C ± 2 DEG C, and 25-35min is kept at this temperature；Then by reactant cooling, ethanol washing, centrifuge separation, institute is finally obtained State extra small ferrite nanometer particle；The presoma, finish, the mass ratio between solvent are 1:0.7-0.8:3-4；

Wherein, the presoma is iron erucic acid complex compound and/or manganese oleic acid complex compound, and the solvent is benzyl oxide, benzyl ether or ten One or more of eight alkene mixture；The finish is one or more of oleic acid, oleyl alcohol or oleyl amine composition.

6. being used for the nano-probe of Alzheimer disease pathogenic protein as claimed in claim 5, it is characterised in that: the presoma It is prepared by the following method: after mixing by salt, erucic acid, sodium hydroxide, being dissolved in methanol, the magnetic agitation at 40 ± 2 DEG C, Reaction 1-2 hours obtains institute in obtained dry 12 hours in 45 ± 2 DEG C of vacuum with deionized water and methanol washed product State iron erucic acid complex compound；The salt, erucic acid, the mass ratio between sodium hydroxide are 1:3-4:0.4-0.5, the salt, acid, hydrogen-oxygen Changing the solid-to-liquid ratio between the mixture and methanol of sodium is 1:10-11g/ml；The salt is iron oxide, iron oleate, erucic acid iron, oleic acid One or more of manganese, erucic acid manganese composition.

7. the preparation method as described in any one of claim 1-6 for the nano-probe of Alzheimer disease pathogenic protein, It is characterised in that it includes following preparation step:

(1) extra small ferrite nanometer particle is added to having in chloroform, and one or more of polyethylene glycol both-end bases is added Substitutive derivative, heating, are stirred to react ultrasound, and organic solvent is removed after reaction；

(2) it is cooled to room temperature, aqueous phase solution is added, dialysis, ultrafiltration after ultrasound, filtering take the extra small iron oxygen of well dispersed water phase Body nanoparticles solution is freeze-dried as powder or is placed in 4 DEG C for use；

(3) phenothiazine derivative is dissolved in dimethyl sulfoxide, it is sub- that 1- ethyl-(3- dimethylaminopropyl) carbon two is added Nano particle obtained in the step of amine, n-hydroxysuccinimide carry out priming reaction, and Aqueous dispersions are then added (2) is molten Liquid reacts 4 hours；

(4) reaction product that step (3) obtains is dialysed in distilled water, the solution after taking dialysis is freeze-dried to get use is stated In the nano-probe of Alzheimer disease pathogenic protein；The molecular cut off of bag filter used is 500-14000Da when dialysis, Sublimation drying is 12-24 hours.

8. the preparation method for the nano-probe of Alzheimer disease pathogenic protein, feature exist as claimed in claim 7 In in the step (1), reaction temperature is 20-70 DEG C, and ultrasonic time is 10-30 minutes, and the reaction time is 2-12 hours, instead Should solvent be removed using rotatory vacuum afterwards.

9. the preparation method for the nano-probe of Alzheimer disease pathogenic protein, feature exist as claimed in claim 7 In in the step (2), ultrasonic time is 10-30 minute, and the filter sizes of filtering are 0.22-0.45 microns, when freeze-drying Between be 12-24 hours.

10. the preparation method for the nano-probe of Alzheimer disease pathogenic protein, feature exist as claimed in claim 7 In, in the step (3), phenothiazine derivative and 1- ethyl-(3- dimethylaminopropyl) carbodiimide and N- hydroxysuccinimidyl Imido mass ratio is 1:2-4:2-6；Nanoparticles solution reaction density is 1-5mg/mL, and nano particle and phenthazine are derivative The mass ratio of object is 1:0.1-2.

Technical field

The invention belongs to nano materials and biomedical molecular image technical field, and in particular to one kind is used for alzheimer ' The nano-probe of silent disease pathogenic protein and its preparation.

Background technique

Alzheimer disease (Alzheimer's disease, AD) is a kind of common dull-witted lesion type, is to carry out Property cognition dysfunction and behavior damage the central nervous system retrogression pathological changes that are characterized.Although having put into both at home and abroad a large amount of Fund carries out the medicament research and development of AD disease, but most of research at present ends in failure 5, wherein most important reason may be Lack effective early diagnosis, the time of therapeutic administratp has been in advanced stage, and medication effect is little.Therefore, research at present The significant challenge of personnel is how to carry out clinicopathological features, the real-time tracking of Alzheimer disease using clinical effective means Pathological change to realize the early diagnosis of Alzheimer disease, and valid prevented and is intervened.

Amyloid protein cascade hypothesis is one of the mainstream theory of current AD pathogenesis, i.e. amyloid precusor protein The beta-amyloid polypeptide (β-Amyloid, A β) of (amyloid peptide precursor, APP), if A β 40, A β 42 are in brain In aggregation, the morbidity of Alzheimer disease is very significant considering that.And the aggregation of the albuminoid usually occurs early in disease Before clinical symptoms, even just had existed for before 20 years.Therefore, diagnosis of Alzheimer disease preparation can be constructed, it is right Such pathogenic protein carry out molecular level label and tracking, be realize early stage diagnosis of Alzheimer disease effective way it One.

Currently, people have carried out a degree of research, such as positron emission computerized tomography in terms of diagnostic preparation (PET) image probe¹¹C-PIB, fluorescence imaging molecule NIAD, Methoxy-X04, AOI-987, CRANAD-2 etc..However, these Marker is coupled with radioactive element, the medication scope of application is limited, long-term follow monitors difficult and diagnosis expense of great number etc. Problem affects its extensive use in terms of the early diagnosis of AD disease.

Summary of the invention

To solve the above-mentioned problems, the purpose of the present invention is to provide a kind of receiving for Alzheimer disease pathogenic protein Rice probe.

The second object of the present invention is to provide the preparation method of the nano-probe for Alzheimer disease pathogenic protein.

The present invention is achieved through the following technical solutions:

A kind of nano-probe for Alzheimer disease pathogenic protein, the nano-probe are multi-modal nano-probe, Kernel is extra small ferrite nanometer particle, and outer layer is that polyethylene glycol segment is coupled phenothiazine derivative；The water of the nano-probe Kinetic diameter is 1-30nm；The nano-probe includes the following components calculated by weight: extra small ferrite nanometer particle 5-20 parts, 1-5 parts of polyethyleneglycol derivative, 0.1-2 parts of phenothiazine derivative.

The present invention selects extra small ferrite nanometer particle, utilizes its outstanding r₁Relaxation rate, building have targeting T₁Energy is imaged The probe of power not only avoids the T of traditional gadolinium₁The potential neurotrosis of contrast medium bring, and its extra small size is also advantageous In penetrating for blood-brain barrier.Meanwhile the coupling and introducing of phenothiazine derivative within the probe, so that probe is had excellent A β and ties up Determine marked capacity and near-infrared fluorescent specificity enhancing ability.Polyethyleneglycol derivative for connect iron oxygen nano particle and Phenothiazines.

Preferably, the phenothiazine derivative structure are as follows:

Wherein, m is the number of double bond, including and be not limited to following number, 1,2,3 etc.；N is alkyl chain number, including and It is not limited to following number, 1,2,3 etc.；R₁It is methoxy base class, carboxyl class, ammonia for the functional group for connecting polyethyleneglycol derivative The molecules such as base class, sulfydryl class, maleimide, nitrine class, biotin or N- succinimido；R₂For modification group, it is The molecules such as hydrogen, hydroxyl, methyl, ethyl, halogen, carboxyl, primary amine groups or tertiary amine groups.

Preferably, the polyethylene glycol is the substitution of both-end base, end group is methoxyl group, amino, sulfydryl, carboxyl, Malaysia acyl Imido grpup or alkynyl.

Preferably, the polyethyleneglycol derivative structural formula are as follows:

Wherein, R₁For connect ferrite nanometer particle functional group, be alkyl, amino, carboxyl, sulfydryl, phosphoric acid salt, The molecules such as hydroxamic acid or catechol；R₂It is methoxy base class, carboxyl for the functional group for connecting phenothiazine derivative The molecules such as class, amino, sulfydryl class, maleimide, nitrine class, biotin class or N- succinimido；N refers to gathering The repetitive unit number of ethylene glycol, according to required molecular weight, n can be from any number of 1-200.

Preferably, the partial size of the extra small ferrite nanometer particle is 1-6nm, the extra small ferrite nanometer particle The preparation method comprises the following steps: being first uniformly mixed presoma with finish, then mixture is dissolved in solvent and being stirred evenly, will be then mixed with The mixture of solvent is heated to 265 DEG C ± 2 DEG C, and keeps 25-35min at this temperature；Then by reactant, cooling, ethyl alcohol is washed It washs, be centrifugated, finally obtain the extra small ferrite nanometer particle；The presoma, finish, the mass ratio between solvent are 1:0.7-0.8:3-4；

Wherein, the presoma is iron erucic acid complex compound and/or manganese oleic acid complex compound, and the solvent is benzyl oxide, benzyl ether Or one or more of octadecylene mixture；The finish is one or more of oleic acid, oleyl alcohol or oleyl amine composition.

The presoma is prepared by the following method: after mixing by salt, erucic acid, sodium hydroxide, it is dissolved in methanol, in Magnetic agitation at 40 ± 2 DEG C is reacted 1-2 hours, obtained in 45 ± 2 DEG C of vacuum with deionized water and methanol washed product Middle drying obtains the iron erucic acid complex compound for 12 hours；The salt, erucic acid, the mass ratio between sodium hydroxide are 1:3-4:0.4- 0.5, the salt, acid, sodium hydroxide mixture and methanol between solid-to-liquid ratio be 1:10-11g/mL；The salt be iron oxide, One or more of iron oleate, erucic acid iron, manganese oleate, erucic acid manganese composition.

The preparation method of nano-probe for Alzheimer disease pathogenic protein, including following preparation step:

(1) extra small ferrite nanometer particle is added to having in chloroform, and it is double that one or more of polyethylene glycol are added End group substitutive derivative, heating, are stirred to react ultrasound, and organic solvent is removed after reaction；

(2) it is cooled to room temperature, aqueous phase solution is added, dialysis, ultrafiltration after ultrasound, filtering take well dispersed water phase extra small Ferrite nanometer particle solution is freeze-dried as powder or is placed in 4 DEG C for use；

(3) phenothiazine derivative is dissolved in dimethyl sulfoxide, 1- ethyl-(3- dimethylaminopropyl) carbon two is added Nano particle obtained in the step of imines, n-hydroxysuccinimide carry out priming reaction, and Aqueous dispersions are then added (2) is molten Liquid reacts 4 hours；

(4) reaction product that step (3) obtains is dialysed in distilled water, take dialysis after solution, freeze-drying to get State the nano-probe for Alzheimer disease pathogenic protein.

Preferably, in the step (1), reaction temperature is 20-70 DEG C, and ultrasonic time is 10-30 minutes, and the reaction time is 2-12 hours, solvent was removed using rotatory vacuum after reaction.

Preferably, the filter sizes of filtering are it is characterized in that, ultrasonic time is 10-30 minutes in the step (2) 0.22-0.45 microns, sublimation drying is 12-24 hours.

Preferably, it is characterized in that, in the step (3), phenothiazine derivative and 1- ethyl-(3- dimethylamino third Base) mass ratio of carbodiimide and n-hydroxysuccinimide is 1:(2-4): (2-6)；Nanoparticles solution reaction density is 1- The mass ratio of 5mg/mL, nano particle and phenothiazine derivative is 1:(0.1-2).Preferably, it is characterized in that, the step (4) in, when dialysis the molecular cut off of bag filter used be 500-14000Da, sublimation drying is 12-24 hours.

The multi-modal nano-probe of targeting provided by the invention includes having the nanoparticles of efficient diagnosis ability and can dividing Scattered physiology aqueous solution.The diagnosis object is wrapped up by extra small ferrite nanometer particle, polyethyleneglycol derivative and the pheno thiophene of coupling Oxazine derivatives are constituted, and phenothiazine derivative has specific donor-acceptor molecular structure, and special with outstanding A beta-aggregation body The affinity of property.The targeting T to the brain A β plaque block of living body AD disease mice model can be achieved₁-T₂Tri- mode of MRI/NIRF at Picture.

The present invention utilizes extra small ferrite nanometer particle, is coupled the near-infrared fluorescent enhancing with A β specificity and sensibility The phenothiazines of performance construct the T that novel extra small multi-modal nano-probe is used to carry out A β label₁-T₂MRI/NIRF at Picture, nano-probe of the present invention have extra small partial size, no gadolinium neurotoxicity, biocompatibility is good, belong to it is radiationless, Non-intrusive type probe carries out the diagnosis of AD disease, provides good solution party for early diagnosis and long-term observation A β plaque block Case.

Compared with prior art, the multi-modal nano-probe of targeting A β of the invention, will target pathogenic protein, T₁-MRI、 T₂- MRI and near-infrared fluorescence imaging tracer are the entirety of set, it can be achieved that causing a disease in the brain of Alzheimer disease mouse model The positioning of albumen patch is imaged, and is imaged using the MRI of high spatial resolution, shows the T of anatomical details₁- MRI axis information, The position of patch in brain is positioned under living body, because it is with high-biocompatibility, internal metabolic capability, there is no multiple comparisons Gadolinium neurotoxicity caused by agent is administered.Therefore, which has widely in terms of the early diagnosis of Alzheimer disease Application prospect.

Detailed description of the invention

Fig. 1 is the transmission electron microscope photo figure of the extra small ferrite nanometer particle of oily phase

Fig. 2 is the transmission electron microscope photo figure of the multi-modal nano-probe of targeting prepared by embodiment 1

Fig. 3 is the hydrodynamics grain size distribution of the multi-modal nano-probe of targeting prepared by embodiment 1

Fig. 4 is the transmission electron microscope photo figure of the extra small ferrite nanometer particle of water phase

Fig. 5 is the hydrodynamics grain size distribution of the extra small ferrite nanometer particle of water phase

Fig. 6 is the staining brain sections NIR fluorescence microscopy figure for targeting multi-modal nano-probe

Fig. 7 is the internal NIR fluorescence imaging figure for targeting multi-modal nano-probe

Fig. 8 is the external NIR fluorescence imaging figure for targeting multi-modal nano-probe

Fig. 9 is the external T for targeting multi-modal nano-probe₁-T₂MRI image

Figure 10 is the internal T for targeting multi-modal nano-probe₁-T₂MRI image

Specific embodiment

The present invention is described in further detail With reference to embodiment, to help those skilled in the art's reason The solution present invention.