CN109045311B

CN109045311B - Prussian blue nano MRI tracer agent and preparation method and application thereof

Info

Publication number: CN109045311B
Application number: CN201810995110.4A
Authority: CN
Inventors: 王金环; 常津; 徐立霞; 罗仲秋; 刘雅娟; 李家林; 王琼
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-08-29
Filing date: 2018-08-29
Publication date: 2021-07-20
Anticipated expiration: 2038-08-29
Also published as: CN109045311A

Abstract

The invention discloses a Prussian blue nano MRI tracer agent and a preparation method and application thereof, and provides a non-toxic Prussian blue nano particle-based MRI tracer agent with good specificity and sensitivity, wherein the Prussian blue tracer agent is combined with maleimide Prussian blue nano particles of beta-amyloid protein 42 modified by cysteine, can detect Abeta deposited in the brain of an Alzheimer patient, and can clearly image on MRI. The invention takes A beta homologous polypeptide as a target of beta amyloid in brain, takes Prussian blue as an imaging agent, and prepares an MRI tracer agent which can pass through a blood brain barrier and can be specifically combined with A beta. The invention can provide a new means for AD early diagnosis and curative effect evaluation as a detection agent of molecular images.

Description

Prussian blue nano MRI tracer agent and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a Prussian blue nano MRI tracer agent, and a preparation method and application thereof.

Background

Clinically, Alzheimer Disease (AD) is mainly caused by intelligent damage, is manifested by senile dementia of middle-aged and elderly people, and seriously threatens the health of middle-aged and elderly people in China. At present, no effective treatment method for AD exists, and with the intensive research, more and more evidences indicate that the accumulation of extracellular amyloid (A beta), the entanglement of intracellular nerve fibers and the loss of neurons are the remarkable pathophysiological characteristics of AD. The major component of amyloid is the enzymatic product of amyloid β -protein precursor protein (APP), which is secreted extracellularly by the cell. Based on the "amyloid hypothesis" of AD pathogenesis, a number of therapeutic approaches targeting a β are under investigation and some have been introduced in clinical trials. From these findings, therapeutic measures for AD often lead to better therapeutic results if intervention can be given early in the disease. At present, the exact diagnosis of AD still depends on the histopathological characteristics of autopsy for definite diagnosis, the structural change of the pathology is often in the middle and late clinical stage, the treatment effect is poor at the moment, and the progress of the disease condition is difficult to reverse, so that the realization of the early diagnosis and the curative effect evaluation of AD patients is a very urgent hotspot problem.

Researchers are working on finding early diagnostic markers and disease repair therapies in an effort to provide direct imaging evidence for early diagnosis of AD. Positron Emission Tomography (PET) based images of Α β deposits have been successfully applied to AD patients. However, because PET-CT has the disadvantages of radioactivity, low spatial resolution, high examination cost, low machine popularity, and narrow application range, researchers are trying to find tracers that are non-radioactive, high in popularity, strong in specificity, and non-toxic, and tracers based on Magnetic Resonance Imaging (MRI) are expected to be an important means for achieving this goal. MRI based on Α β deposition scans brain tissue from dead AD patients to see individual Α β plaques on the T2 weighted image, but scan times as long as 20 hours, and are not useful for in vivo studies, and MRI detection is not very specific to Α β, and it is also possible to detect other structures high in iron content, such as old bleeding calcifications remaining in the brain. Prussian blue is an antidote approved by FDA and applied to clinically treating thallium and other radioactive element toxicities, is a traditional nano material, mainly consists of a ferroferric oxide nano core and a Prussian blue nano shell, is an efficient near-infrared photothermal agent, and has good biocompatibility and biological safety. Ferroferric oxide has good nuclear magnetic resonance imaging and magnetic targeting effects and is also applied to clinic. However, the use of magnetic prussian blue nanoparticles as a material for targeted diagnosis of alzheimer has not been reported.

Disclosure of Invention

We need a specific and highly specific tracer to select for a β senile plaques to increase the sensitivity of the method in the hope of achieving a definitive diagnosis of a β deposition during the early stages of AD. However, at present, no specific nuclear magnetic tracer agent targeting at Abeta is available at home, and the clinical diagnosis of AD at home lacks direct early evidence, so the invention aims to provide a method for preparing a nuclear magnetic resonance tracer agent with no toxicity, good specificity and sensitivity, can detect the Abeta deposited by AD, and provides a new method for early diagnosis and curative effect evaluation of AD in future.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of Prussian blue nano MRI tracer agent comprises the following steps:

1) preparation of cysteine-modified beta-amyloid 42

Cysteine-modified beta-amyloid 42 is synthesized by inserting cysteine containing a thiol group between lysine at position 28 and glycine at position 29 of beta-amyloid 42, and the sequence thereof is: DAEFRHDSGYEVHHQKLVFFAEDVGSNK(Cys) GAIIGLMVGGVVIA are provided.

2) Preparation of Maleiminated Prussian blue nanoparticles

Dropwise mixing FeCl 3.6H2O deionized water mixed solution of 0.5mM citric acid and 1mM K4Fe (CN)6 deionized water mixed solution of citric acid at 60 ℃, continuously stirring for 5min, adding 0.005mM polyetherimide solution, continuously stirring for 4 hours, then centrifugally separating, collecting precipitate, washing with acetone and deionized water, and carrying out heavy suspension with dimethyl sulfoxide to obtain aminated Prussian blue nano-particles;

adding a dimethyl sulfoxide solution of maleic anhydride into the prepared aminated Prussian blue nanoparticle solution, continuously stirring for 1-3 days, centrifugally separating, washing and re-suspending to obtain a solution A; adding N- (2-hydroxyethyl) maleimide N, N-dimethylformamide solution into the solution A, continuously stirring, centrifugally separating, washing, and resuspending with phosphate solution to obtain maleimide Prussian blue nanoparticles;

3) preparation of Maleiminated Prussian blue nanoparticles incorporating cysteine-modified beta-amyloid 42

Mixing the cysteine-modified beta-amyloid 42 prepared in the step 1) and the maleimide-modified prussian blue nanoparticles prepared in the step 2) in a mass ratio of 8: 1-12: 1, uniformly mixing and carrying out suspension culture at 4 ℃ for 2-6h, centrifuging, washing, finally carrying out heavy suspension by using a phosphate solution, and placing the mixture in a refrigerator at 4 ℃ for standby application to obtain the maleimide Prussian blue nano-particles combined with the cysteine modified beta-amyloid protein 42.

Further, the sequence of the cysteine-modified beta-amyloid 42 in the step 1) is the sequence shown in SEQ ID NO. 1.

Further, the mass ratio of the cysteine-modified β -amyloid 42 to the maleimidoylated prussian blue nanoparticles in step 3) is 10: 1.

the invention also aims to provide the Prussian blue nano nuclear magnetic tracer.

The surface of the nano nuclear magnetic tracer is negatively charged, particles are of a cubic structure, and the particle size is 60-70 nm.

The invention also aims to provide application of the Prussian blue nano nuclear magnetic tracer in preparation of a preparation for diagnosing the Alzheimer disease.

Advantageous effects

The invention provides a non-toxic Prussian Blue (PB) nanoparticle-based MRI tracer agent with good specificity and sensitivity, and maleimide Prussian blue nanoparticles (PB @ PEI @ MAL-cys-Abeta 42) combined with cysteine-modified beta-amyloid 42 can detect Abeta deposited in the brain of an Alzheimer patient and clearly image on MRI. The invention takes A beta homologous polypeptide as a target of beta amyloid in brain, takes Prussian blue as an imaging agent, and prepares an MRI tracer agent which can pass through a blood brain barrier and can be specifically combined with A beta. The invention can provide a new means for AD early diagnosis and curative effect evaluation as a detection agent of molecular images.

1. The nuclear magnetic tracer PB @ PEI @ MAL-cys-Abeta 42 NPs has good stability, so that the nuclear magnetic tracer PB @ PEI @ MAL-cys-Abeta 42 NPs can be transported for a long distance and stored for a long time.

2. The PB @ PEI @ MAL-cys-Abeta 42 NPs prepared by the invention selects a femoral intravenous injection mode, and is safe and convenient in clinical application.

3. The PB @ PEI @ MAL-cys-Abeta 42 NPs prepared by the invention has good biocompatibility and biological safety, and has no toxicity to cells.

4. The PB @ PEI @ MAL-cys-Abeta 42 NPs prepared by the invention can identify senile plaques in the brain, and has good specificity and sensitivity.

Drawings

FIG. 1 is a flow chart of the preparation process of PB @ PEI @ MAL-cys-Abeta 42 NPs in example 1;

FIG. 2 is a TEM image of PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1;

FIG. 3 is a particle size plot of PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1; wherein, FIG. 3a is the particle size distribution of PB @ PEI @ MAL-cys-Abeta 42 NPs, and FIG. 3b is the potential characterization of PB @ PEI @ MAL-cys-Abeta 42 NPs;

FIG. 4 is an IR spectrum of PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1;

FIG. 5 is a graph of the relaxation times T1, T2 as a function of concentration for the MRI of PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1;

FIG. 6 is a graph of the affinity assays for different concentrations of PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1;

FIG. 7 is a cytotoxicity assay of PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1;

FIG. 8 is a graph of the MRI T2 weighted images of the PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1 on APP/PS1 AD transgenic mice; wherein FIG. 8a is a littermates group and FIG. 8b is an APP/PS1 AD mouse group.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings and the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by those skilled in the art without any creative work belong to the protection scope of the present invention.

Example 1

1) Preparation of cysteine-modified beta-amyloid 42 (cys-Abeta 42)

2) Preparation of Maleiminated Prussian blue nanoparticles (PB @ PEI @ MALNPs)

Mixing solution A was prepared by dissolving 0.5mM citric acid in 1mM FECL 3.6H 2O DI water, mixing solution B was prepared by dissolving 0.5mM citric acid in 1mM K4Fe (CN) 6.3H 2O DI water, and 20ml of mixing solution B was added dropwise to 20ml of mixing solution A at 60 deg.C with stirring for 5min until the mixing solution became a bright dark blue color. Adding a Polyetherimide (PEI) solution with the concentration of 0.005mM, continuously stirring for 4h at 60 ℃, centrifuging at 15000rpm for 30min, collecting precipitates, washing with acetone and deionized water, and finally resuspending with dimethyl sulfoxide to obtain the aminated Prussian blue nanoparticles (PB @ PEI NPs).

Preparing maleic anhydride into a 0.05-0.2 mM solution by using dimethyl sulfoxide, adding the solution into the prepared solution, continuously stirring for 1-3 days, collecting a mixed solution, centrifuging for 30min at 15000rpm, washing the precipitate for 2-3 times by using deionized water, and finally resuspending the precipitate by using N, N-dimethylformamide to obtain a solution C. Preparing N- (2-hydroxyethyl) maleimide into a 0.2-0.5 mM solution by using N, N-dimethylformamide, adding the solution into the solution C, continuously stirring for 1-3 days, collecting mixed solution, centrifuging at 15000rpm for 30min, washing the precipitate with deionized water for 2-3 times, and finally suspending with a phosphate solution to obtain the maleimide Prussian blue nanoparticles (PB @ PEI @ MAL NPs).

3) Preparation of Maleiminated Prussian blue nanoparticles incorporating cysteine-modified beta-amyloid 42 (PB @ PEI @ MAL-cys-A β 42 NPs)

Dissolving the maleimide prussian blue nanoparticles prepared in the step 2) in 4ml of PBS, and then mixing the cysteine-modified β -amyloid 42 prepared in the step 1) and the maleimide prussian blue nanoparticles prepared in the step 2) (the mass ratio of the cysteine-modified β -amyloid 42 to the maleimide prussian blue nanoparticles is 10: 1) adding into the above solution, mixing and suspending at 4 deg.C for 2-6h, centrifuging, washing, suspending with phosphate solution, and storing in refrigerator at 4 deg.C for use to obtain cysteine modified beta-amyloid protein 42-combined maleimide Prussian blue nanoparticles (PB @ PEI @ MAL-cys-A beta 42 NPs).

Further, the sequence of cys-A beta 42 in the step 1) is the sequence shown in SEQ ID NO. 1.

Example 2

The Prussian blue nano nuclear magnetic tracer PB @ PEI @ MALCys-A beta 42 NPs prepared in the example 1 is detected and characterized.

FIG. 2 is a transmission electron micrograph of Prussian blue nano nuclear magnetic tracer PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1

The method comprises the following steps of carrying out appearance characterization on PB @ PEI @ MAL-cys-Abeta 42 NPs through a transmission electron microscope, and specifically comprising the following operation steps:

dissolving PB @ PEI @ MAL-cys-Abeta 42 NPs with PBS, ultrasonically dispersing, sucking 10 mu l of the solution, dropwise adding the solution onto a copper net covered with an ultrathin carbon film, fully adsorbing for 10-30min until a sample is dried, observing in a transmission electron microscope, and taking a picture. As shown in FIG. 2, it can be seen that PB @ PEI @ MAL-cys-Abeta 42 NPs have a cubic structure with a particle size of about 60nm, uniform particle size distribution and good dispersibility.

FIG. 3 is a particle size diagram of Prussian blue nano nuclear magnetic tracer PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1

Wherein FIG. 3a is a particle size distribution of PB @ PEI @ MAL-cys-Abeta 42 NPs and FIG. 3b is a potential characterization of PB @ PEI @ MAL-cys-Abeta 42 NPs.

The particle size distribution and potential characterization of PB @ PEI @ MAL-cys-Abeta 42 NPs are measured by a laser particle sizer by means of a dynamic light scattering method, and the specific operation steps are as follows:

after PB @ PEI @ MAL-cys-Abeta 42 NPs are ultrasonically dissolved and dispersed by PBS, 2-3mL of the mixture is placed in a quartz cuvette, then dynamic light scattering particle size determination is carried out, and each sample is repeatedly measured for 3 times to obtain an average value. The particle size of the Prussian blue nano nuclear magnetic tracer shown in figure 3 is about 65nm, and the surface of the Prussian blue nano nuclear magnetic tracer carries negative charges.

FIG. 4 is an infrared spectrum of Prussian blue nano nuclear magnetic tracer PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1

The method is characterized by measuring the absorption spectrum of PB @ PEI @ MAL-cys-Abeta 42 NPs particles by an ultraviolet-visible spectrophotometer, and comprises the following specific operation steps:

carrying out ultrasonic dispersion on PB @ PEI @ MAL-cys-Abeta 42 NPs by using a PBS solution, placing 2-3mL of the dispersed product in a quartz cuvette, carrying out zero calibration by using deionized water, scanning and observing results in a wavelength range of 350-900nm, and drawing an absorption curve, wherein the scanning interval is 2 nm. As can be seen in FIG. 4, the prepared Prussian blue nano nuclear magnetic tracer PB @ PEI @ Mal-cys-Abeta 42 NPs has a relatively strong and wide absorption peak in a near infrared light region and is strongest at about 720 nm.

Example 3

FIG. 5 is a graph of the concentration-dependent changes of relaxation times T1 and T2 in MRI of Prussian blue nano-nuclear magnetic tracers PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1

Preparing a PB @ PEI @ MAL-cys-Abeta 42 NPs solution with a concentration gradient of 0-16mg/ml by using PBS, and uniformly dispersing the solution in a nuclear magnetic sample tube; the spin-lattice relaxation time (T1) and the spin-spin relaxation time (T2) of each concentration gradient solution were measured by a 1.5T nuclear magnetic resonance analyzer, while taking grayscale images. FIG. 5 shows that relaxation times T1 and T2 of weighted nuclear magnetic resonance imaging of Prussian blue nano nuclear magnetic tracers PB @ PEI @ MAL-cys-Abeta 42 NPs are increased along with the increase of the concentration of PB @ PEI @ MAL-cys-Abeta 42 NPs in a solution.

Example 4

Affinity detection is carried out on the Prussian blue nano nuclear magnetic tracer PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in the example 1.

The affinity of PB @ PEI @ MAL-cys-Abeta 42 NPs and Abeta polypeptide is detected by an ELISA method, and the specific operation steps are as follows:

mu.l of A.beta.40 (1. mu.g/mL) was added to each well of a 96-well polystyrene microplate (2HB), and the mixture was shaken overnight at 4 ℃. Blocking (TBS-T-1% BSA) at room temperature for 2 hours, diluting PB @ PEI @ MAL-cys-Abeta 42 NPs with blocking buffer, adding 50. mu.l of sample per well, and performing shaking reaction at room temperature for 2 hours. Add 50. mu.l/well of primary anti-R422 capable of detecting only A.beta.42 (1:500, this antibody cannot detect A.beta.40), shake at room temperature for 3 hours, add 50. mu.l of anti-mouse horseradish peroxidase antibody (1:1000) per well after washing, shake at room temperature for 1 hour, add 50. mu.l of TMB per well after washing, shake until the color changes, add 20. mu.l of 2N H2SO4 per well to stop the reaction, and read the plates at a wavelength of 450 nm. The results in FIG. 6 show that A β 40 has a good binding force with PB @ PEI @ MAL-cys-A β 42 NPs.

Example 5

The Prussian blue nano nuclear magnetic tracer PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in the example 1 is used for in vitro detection of cytotoxicity

The cytotoxicity of PB @ PEI @ MAL-cys-Abeta 42 NPs is determined by adopting an MTT cell proliferation test, and the specific operation steps are as follows:

human neuroblastoma cell line (SK-N-SH) was seeded at 10,000 cells per well in 96-well flat-bottom plates, incubated overnight at 37 ℃ with 5% CO2, followed by the next day with different doses of PB @ PEI @ MAL-cys-Abeta 42 NPs, followed by 72 hours with 10. mu.l of 5mg/ml MTT per well, incubation continued for 4 hours, and finally the medium was aspirated off, 150. mu.l DMSO was added, and the plates were read at 490 nm. FIG. 7 shows that PB @ PEI @ MAL-cys-Abeta 42 NPs have extremely low cytotoxicity to cells, and can still keep the cell survival rate to be more than 85% at the highest concentration, thus proving that the cell biological safety of the nano composite particles is good.

Example 6

Carrying out in vivo nuclear magnetic imaging on the Prussian blue nano nuclear magnetic tracer PB @ PEI @ MAL-cys-Abeta 42 NPs prepared in example 1

APP/PS1 AD transgenic mice of 12 months and littermates of littermates are selected as detection models. We injected PB @ PEI @ MAL-cys-abeta 42 NPs (10mg/ml, 100 μ l) via the left femoral vein using a PHD2000 injection pump, examined by MRIT2 weighted imaging at 8 hours post-injection, and the nuclear magnetic MRIT2 weighted image in fig. 8b showed that the hippocampus and the plateau regions of APP/PS1 AD transgenic mice brain showed a large number of dark spots, i.e., imaging of intracerebral abeta amyloid on MRI, whereas the litters group in fig. 8a did not detect, indicating that the tracer PB @ PEI @ MAL-cys-abeta 42 NPs was able to specifically detect senile plaques in AD transgenic mice.

The invention provides an application of a Prussian blue nano nuclear magnetic tracer in preparation of a preparation for diagnosing Alzheimer's disease, in particular to a nuclear magnetic contrast agent specifically targeting senile plaques in brain.

Sequence listing

<110> Wangjin ring

<120> Prussian blue nano MRI tracer agent and preparation method and application thereof

<141> 2018-08-27

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 43

<212> PRT

<213> cysteine-modified beta-amyloid 42(2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 1

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Cys Gly Ala Ile

20 25 30

Ile Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

<210> 2

<211> 0

<212> PRT

<213> cysteine-modified beta-amyloid 42(Amylocarpus encephaloides)

<400> 2

Claims

1. A preparation method of Prussian blue nano MRI tracer agent is characterized by comprising the following steps:

1) preparation of cysteine-modified beta-amyloid 42

Cysteine-modified beta-amyloid 42 is synthesized by inserting cysteine containing a thiol group between lysine at position 28 and glycine at position 29 of beta-amyloid 42, and the sequence thereof is: DAEFRHDSGYEVHHQKLVFFAEDVGSNK(Cys) GAIIGLMVGGVVIA, respectively;

2) preparation of Maleiminated Prussian blue nanoparticles

adding a dimethyl sulfoxide solution of maleic anhydride into the prepared aminated Prussian blue nanoparticle solution, continuously stirring for 1-3 days, centrifugally separating, washing, and re-suspending to obtain a solution A; adding N- (2-hydroxyethyl) maleimide N, N-dimethylformamide solution into the solution A, continuously stirring, centrifugally separating, washing, and resuspending with phosphate solution to obtain maleimide Prussian blue nanoparticles;

2. The method for preparing prussian blue nano MRI tracer according to claim 1, wherein the sequence of the cysteine-modified β -amyloid 42 in step 1) is the sequence shown in SEQ ID No. 1.

3. The method for preparing prussian blue nano MRI tracer according to claim 1, wherein the mass ratio of the cysteine-modified β -amyloid 42 to the maleimidoylated prussian blue nanoparticles in step 3) is 10: 1.

4. a prussian blue nanommri tracer prepared according to the method of claim 1.

5. The Prussian blue nano MRI tracer agent according to claim 4, wherein the Prussian blue nano MRI tracer agent has a negatively charged surface, and has a cubic structure and a particle size of 60-70 nm.

6. Use of a prussian blue nanommri tracer prepared according to the method of claim 1 for the preparation of a formulation for the diagnosis of alzheimer's disease.