Discovery Of Novel Theranostic Agent For Both Aβ Diagnosis And Therapy

Alzheimer’s disease（AD）is a neurodegenerative disease with cognitive impairment as its main clinical manifestation.At present,drug development based on theβ-amyloid（Aβ）hypothesis has failed,which may be attributed to the following two reasons:one is that the intervening time may be too late,as the Aβplaque has already formed and the toxic form of Aβhas already caused nerve damage at that time;the other is that abundant soluble Aβoligomers existed before the deposition of Aβfibrils is increasingly believed as the most toxic form of Aβ.Therefore,the early diagnosis of targeted Aβis of great significance for the treatment of AD.On the other hand,the effective inhibition of the formation and toxicity of Aβoligomers is an important and alternative way to treat AD.Recently,the concept of"theranostic"with dual functions of diagnosis and treatment has been derived as molecular diagnostic probes and therapeutic drugs may acting through the same key target,and has already shown good prospects in the field of neuropharmacology in recent years.Taking Aβ,the key toxic polypeptide for early diagnosis and pathogenesis of AD,as the key target,this thesis studies novel high-efficiency near-infrared fluorescent imaging probes for Aβ.Based on this,we further study a theranostic fluorescent probe and a fluorescent probe/material complex which can effectively inhibit the aggregation and toxicity of Aβ.Moreover,the binding and imaging mechanisms of theranostic fluorescent probe towards different Aβforms are further studied.In the first part of this thesis,QAD-1,a novel“off-on”near-infrared fluorescent probe,was obtained through rational design,synthesis and biological research.Firstly,the near infrared fluorescent imaging molecule QAD-1 with quenched group was rational designed and synthesized.It was confirmed that the near infrared fluorescence value of QAD-1 with quenched group was significantly reduced and had off effect at the in vitro level.We further confirmed the“off-on”near-infrared fluorescent imaging effect of QAD-1 at molecular,brain slice and animal levels:at the in vitro level,it was found that the fluorescent signal of QAD-1 was enhanced with the increase of the concentration of Aβfibrils,and the K_d value of binding to Aβfibrils was 27 nM;at the brain slice level,it was found that the co-incubation of QAD-1 could specifically identify Aβplaques,and the results of near infrared Aβimaging without washing procedure showed high signal-to-noise ratio,which is superior to the fluorescent imaging of ThS,a traditional push-pull fluorescent molecule that require washing procedure.At the level of 15-month-old AD transgenic animals with large amount of Aβplaques,intravenous administration of QAD-1 has the ability of rapid imaging and rapid brain clearance.Further research in this thesis suggests that QAD-1 has the ability of early diagnose.In AD patients,Aβis first excessively produced,then aggregated into oligomers,and finally fibrosis Aβis deposited into senile plaques in the brain.Therefore,fluorescent probes recognize different forms of Aβand high sensitivity of Aβfibril recognition may have early diagnostic effect.Our results showed that the pattern of QAD-1 staining on brain sliced of AD transgenic mice was similar to that of Aβantibody staining,and the staining range was wider than that of ThS staining,which selectively recognized Aβfibrils.Moreover,besides the high binding affinity of QAD-1 to Aβfibrils,the K_d values of QAD-1 binding to Aβmonomers and oligomers have also reached nM level.On this basis,the brain slices and animal levels of 6-month-old AD transgenic animals with sporadic senile plaques confirmed that QAD-1 has the ability of near infrared fluorescent imaging of Aβ.These results suggest that QAD-1 is an“off-on”near-infrared fluorescent probe of Aβin vivo with Aβearly diagnostic ability.On the basis of QAD-1,the active molecule P14 with dual potential of diagnosis and treatment of Aβwas further developed.In this thesis,a novel theranostic molecule P14 was designed and synthesized by combining the diagnostic ability of QAD-1 and the antagonistic effect of QAD-1 structural analogue compound 4 on the aggregation and toxicity of Aβ.The diagnostic effect of P14 on Aβ,the intervention of Aβcascade reaction and the theranostic effect targeted Aβcascade reaction were further systematically studied.In the aspect of diagnostic ability,P14 has the ability of near infrared fluorescent imaging of Aβby molecular,brain slice and animal levels.Further study on fluorescent imaging mechanism revealed that P14 had different fluorescent response ability with different Aβfragments(Aβ_1-17,Aβ_25-35,Aβ_1-42,etc.),suggesting that P14 might play a fluorescence enhancement role by acting with the C-terminal of Aβ.It was found that two important types of Aβin senile plaque,Aβ_1-42 and Aβ_1-40,could be co-localized.Moreover,the fluorescent imaging mode of Aβplaques in the brain by P14 was similar to that of ThT,which mainly recognized Aβin fibrosis state.In the aspect of interfering with Aβcascade,the therapeutic ability and mechanism of P14 were systematically studied at three levels:molecular,cellular and AD transgenic animals.At the molecular level,we first found that P14 can effectively inhibit the ThT fluorescence enhancement induced by Aβfibrils,suggesting that P14 may reduce the formation of Aβfibrils.Further electron microscopic studies showed that the co-incubation of P14 and Aβmonomer could effectively inhibit the formation of Aβfibrils.Co-incubation of P14 with Aβaggregates/fibrils could effectively reduce the degree of Aβaggregation,suggesting that P14 could inhibit the aggregation of Aβand can depolymerize Aβfibrils.At the cellular level,P14 was found to significantly alleviated the decrease of neuronal cell viability and loss of neurites induced by Aβoligomers.At the level of AD transgenic animals,two different strains of AD model animals were administered for one month and three months respectively.The therapeutic ability of P14 was studied from the level and status of Aβin brain,the number and function of neurons,cognitive and psycho-behavioral levels.It was found that P14 could significantly reduce Aβplaques in cerebral cortex and hippocampus in both AD model animals,suggesting that P14 may affect the number of Aβplaques by influencing the aggregation of Aβ,but not through reducing Aβlevels.P14 significantly alleviated the loss of neurons and the decrease of synaptic protein PSD-95 in the brains of AD mice,but had no significant effect on the inflammatory response of glial cells.In additions,the administration of P14 could significantly ameliorate the cognitive and behavioral impairment of the channel maze in AD model animals.After all these studies,we evaluated the theranostic ability of P14.Using P14 as a diagnostic reagent,it was found that the near-infrared fluorescent signal in P14-treated AD transgenic animals was significantly lower than that in vehicle-treated AD transgenic animals,suggesting that long-term administration of P14 could reduce the level of Aβin the brain and possess the theranostic ability on AD transgenic mice.In the second part of this thesis,based on the limitation of the traditional push-pull fluorescent molecule YHY-1,a new type of"D-A-D"fluorescent probe YHY-2 with high signal-to-noise ratio was developed.YHY-2 was demonstrated to possess Aβ-specific near-infrared imaging ability at three levels:molecule,AD animal brain slice and AD animal.Secondly,based on the characteristics of the new AIE-type Aβfluorescent probe EDS which recognizes the aggregation state of the target,the EDS/MoS₂ complex is formed by combining graphene-like materials with photothermal ability.In terms of diagnosis,EDS/MoS₂ complex enhanced the fluorescent signal of the fluorescent probe when binding to Aβ.In terms of treatment,EDS/MoS₂ complex depolymerized senile plaques in the brain slices of AD transgenic mice under light irradiation.These results suggest that EDS/MoS₂ complex has dual potential of"diagnosis"and"treatment".In this thesis,two kinds of near infrared fluorescent imaging molecules,“off-on”near infrared fluorescence probe QAD-1 and“D-A-D”near infrared fluorescence probe YHY-2,have been investigated.Furthermore,two kinds of fluorescent molecules and fluorescent molecule/photothermal material complex with dual potential of diagnosis and treatment,near infrared fluorescent molecule P14 and“AIE”near infrared fluorescent molecule/MoS₂ complex,have been developed.Moreover,the mechanism of P14 fluorescent imaging and the ability of differently recognizing Aβ_1-40 and Aβ_1-42were preliminarily revealed,which provided a new candidate for precise Aβdiagnosis and a new clue a for simultaneous diagnosis and treatment of AD.