Manufacture Of Multifunctional/Targeting Fe₃O₄ Nano Molecular Probe And Its Application In Imaging And Photothermal Therapy To Alzheimer's Disease

Alzheimer's disease is a class of neurodegenerative lesions.Since1901 when the first case was discoveried,the incidence of the disease is increasing year by year and the disease epidemic has a younger trend.The pathology of the disease is the progressive disappearance of neuronal cells,brain tissue atrophy,brain tissue amyloid protein-??A??fibrosis,aggregation and plaque formation and Tau protein over Phosphorylation of neurofibrillary tangles?NFT?in the hippocampus and cerebral cortex.Since the onset of Alzheimer's disease,there have been many hypotheses about the etiology of Alzheimer's disease.The amyloid cascade hypothesis is the most important hypothesis.A variety of studies about genes,pathology,molecular biology and so on provided some proofs to this hypothesis.Based on these researches,most of the strategies for the diagnosis and treatment of Alzheimer's disease were targeted at A? protein and had made great progress.Objective:Based on the comprehensive analysis of the literatures,the purpose of this study is to use Fe₃O₄ nanoparticles which can cause MRI imaging,poly dopamine used by PTT and Congo red of targeting A?protein polymer to build a targeted/multifunctional nano molecular probes and are used in the treatment of Alzheimer's disease.To open up a new perspective on the treatment of Alzheimer's disease and lay the scientific basis and theoretical basis of the PTT of Alzheimer's disease.Method and Materials:1.The first part: The Conge Red/PEG-PDA@Fe₃O₄ nanometer molecular probe was manufactured by high temperature thermal decomposition method,emulsification method and chemical bonding method.The results were characterized by transmission electron microscopy,dynamic light scattering,infrared and ultravioletspectrophotometer and 1.5T MRI.Size,morphology,functional groups,and magnetic properties of the nanometer molecular probe were analyzed.2.The second part: The efficiency and influencing factors of PTT under near infrared light irradiation in vitro of nanometer molecular probes were measured by hot dipole detector.At the same time,the ability of the nanomolecular probe to depolymerize A? protein polymer under near infrared light irradiation in vitro was observed used by atomic force microscope compared the control group without near infrared light irradiation.Furthermore,the targeted ability of nanoprobe with A?protein polymer was tested by UV spectrophotometry.And then the A?protein aggregates were synthesized by copper ion and acidic environment.Under the irradiation of near infrared light,the nanometer molecular probe depolymerized the polymer of A? protein observed by atomic force microscopy.3.In the third part of this study,the cytotoxicity of nanomolecular probes and A? protein polymer to Neuro-2a cells was tested by CCK8 method.In the same time,the detoxification effect of nano-molecular probes under the irradiation of near infrared light were verified by CCK8.In order to further verify the toxicity of nanomolecular probes to organ and organ function of living animals,in the study KM mices injected nano-molecular probes were used to verify the toxicity of nanomolecular probes.After 30 days of injection,blood routine,biochemical tests and pathological test were carried out.Furthermore,the concentration of nano-molecular probe solution suitable for intravenous injection was screened by erythrocyte hemolysis.In addition,in this part,MRI scan was used to observe the changes in the liver signal and ICP analysis wase used to detect the changes in iron elements of some organs,which be used to further study the metabolic distribution of the nano-molecular probe in vivo.To achieve the nano-probe targeting A? protein polymer on the brain,the probe must have ability of penetrating the blood-brainbarrier permeability.In this study,the nano-molecular probes were used to incubate together with the brain microvascular cells.After the cells were washed,the changes of the signals of the brain microvascular cells were observed by the MRI.The purpose of constructing nanoprobe is to achieve magnetic resonance imaging of A? protein.Therefore,the Conge Red/PEG-PDA@Fe₃O₄ was dispersed in 0.3ml deionized water containing 15% mannitol and injected into the transgenic mice by tail vein injection at a dose of 200?mol Fe/kg,At the time of 0,2,4,6,8,12,24 and 48 hours after injection,4.7T MRT T2*WI scan was performed to observe the signal changes of MRI images in the brain and Z score was used to evaluate the contrast noise ratio of images.At the same time,transgenic mouse brains were acquired after 12 hours injection of nanomolecular probe of and then were used to do pathological Prussian blue?nucleus solid red?staining.At the end of this study,AD mice were treated with 808 nm near-infrared laser 2W/cm2 radiation dose for 7minutes once every other day for 30 days.At the same time,KM rats were taken as blank control group and AD rats with injected nano-probe were taken as negative control group.And then morris water maze experiment was used to test the ablitiy of learning and remembering the space identification,which was verified by Congo red staining.Results:1.The results of the first part showed that the basic average particle size of the Conge Red/PEG-PDA@Fe₃O₄ was 40 nm and the thickness of the dopamine coating was about 5nm.The average hydrated particle size of the nanometer molecular probe in the colloid was about 50 nm,and the particle size was not significantly increased compared with the original particle size of the nanoparticles.The distribution of the nanoparticles was higher.The Zeta potential of the nanoparticles is-30 mv,which demonstrated that there was a good repulsive force between the nanoparticles which can be well dispersed.Infrared and ultravioletspectrophotometers demonstrate that Congo Red/PEG was conjugated to the surface of PDA@Fe₃O₄ nanoparticles and the loading rate of Congo red was.By obtaining the T2 Mapping magnetic resonance image and measuring the T2 value of the nano-molecular probe solution with different Fe content,the T2 relaxation rate of the nanometer molecular probe was 106.2m M-1S-1,which indicates that the nanometer probe has good paramagnetic function.2.The results of the second part showed that the temperature of the nanometer molecular probe rises rapidly in a short time under the irradiation of near infrared light,and the nanometet had the photothermal character.Thermal conversion rate was decided by the concentration and radiant energy.By analyzing the UV spectra of Conge Red/PEG-PDA@Fe₃O₄ + A?,Conge Red/PEG-PDA@Fe₃O₄ and A?proteins,it was found that the UV spectra of nanomolecular probes has a unique peak compared those of the molecular probe and A? protein.The changes in the displacement and response of the peak indicate that the nanomolecular probe can target to the A? protein polymer.Compared with the control group,the shape of the A? protein polymer has significantly changed.3.The results of the third part showed that the nanomolecular probe did not affect the cell viability of Neuro-2a cells.While the A? protein polymer resulted in a 40% decrease in cell viability,however the cell viability increased to 85% under the nanomolecular probe and near infrared light.The above results indicated that nanomeric probes were safe at the cellular level and at the same time reduced the toxicity of A?protein to cells.In the cytotoxicity test,there was no significant change in the blood routine,biochemical tests between the experimental group and control group.There were no organ damage of liver,spleen,lung,kidney and brain in the experimental group.And 1mg/m L nanomolecular probe can be safely used for the intravenous injection.In vivo metabolicexperiments,the liver signal was significantly decreased after 4h injection of nanomolecular probe,and the signal intensity of liver was the lowest at24 hours after injection and reached the peak.The signal intensity of liver was gradually increased at 72 h and 90 h.And there was no significant difference in liver signal intensity between the 90 h group and the blank control group,which indicated that the nano-molecular probe metabolized out from the liver.ICP test has the same experimental results to the MRI.The nano-molecular probe metabolism in the brain reached the peak after 12 hours injection of nanomolecular probe.After incubation,the nanoprobe entered into the inner of the brain microvascular cells and decreased the magnetic resonance signal.In MRI imaging,there were many point-like signal reduction areas in the T2*WI image of the cortex and hippocampus after 12 hours of injection of the nanoprobe.The Z score showed that the image had better contrast noise ratio.There was a statistically significant difference of the Z score betweent the imaging of pre-injection and injection.According to the Prussian blue?nucleus solid red?staining it is confirmed that the presence of nanomeric probes in brain tissue.The results of the treatment showed that the ability of locating navigation and remembering the spatial identification of AD mices were improved.Pathological Congo red showed that the number of A? protein polymer in the brain of AD mice treated with photothermia was reduced and the shape had become smaller.Conclusions:1.The target/multifunctional Conge Red/PEG-PDA@Fe₃O₄nanomolecular probe was successfully manufactured in this study.2.Conge Red/PEG-PDA@Fe₃O₄ nanomolecular probe has the ability to target A? protein and achieve magnetic resonance imaging in vivo.3.Conge Red/PEG-PDA@Fe₃O₄ nanometer molecular probe with irradiation of near infrared light in vitro can depolymerize A? protein polymer and improve AD transgenic mice spatial learning and memory ability.