Design And Synthesis Of Amyloid-β Species Targeted Molecular Probes For In Vivo Imaging

Alzheimer’s disease(AD)is a neurodegenerative disease,with an insidious onset,long course and complicated etiology.Its main clinical symptoms are cognitive impairment and irreversible memory decline,which seriously affects patients’ quality of life and brings serious economic burden to patients’ families and society.The main pathological features of AD are the deposition of β-amyloid proteins(Aβ proteins),the formation of neurofibrillary tangles(NFTs)and the neuronal loss and dysfunction in the brain of patients.Thus,the early detection of these pathological characteristics is of great significance for accurate diagnosis and timely intervention treatment of AD.In order to promote the early definite diagnosis of AD,a large number of fluorescent probes targeting Aβ species have been developed to realize highly sensitive and specific detection of Aβ species.However,to some extent,the formerly reported fluorescent probes still have the following deficiencies including:1)The fluorescent emission wavelength of the probes after binding with Aβ species is relatively short,and even fall out of the near-infrared region(λem>650 nm),which will lead to shallow tissue penetration depth and be easily interfered by the autofluorescence of tissues,thus reducing the sensitivity and resolution to detect Aβ species;2)Some probes show slow binding kinetics to Aβ species(incubation time>15 min)and moderate binding affinity to Aβ species;3)Moreover,most of the reported fluorescent probes respond with Aβ aggregates in the later stage of AD,instead of Aβ monomers or oligomers in the early stage of AD,which is not conducive for early detection of AD.Therefore,in order for highly sensitive and specific detection of Aβ species in vivo,it is absolutely essential to develop near-infrared fluorescent probes with more red-shifted emission wavelength to monitor Aβ monomers,oligomers and aggregates with rapid binding kinetics,high sensitivity and specificity.In the thesis,for one thing,we reasonably designed and developed a series of near-infrared fluorescent probes with Donor-Acceptor-Donor(D-A-D)architecture for specific binding with and sensitive fluorescence imaging of Aβ species in the brain of AD mice model in vivo.For another thing,based on the work of NIR fluorescence probes,an Aβ species responsive NIR fluorescence/positron emission computed tomography(PET)bimodal probe was developed via radio-labelling of 68Ga to fluorescence probe,aimed at highly sensitive and specific detection of Aβ species in vivo.The contents are as follows:In the first part of the paper,probe 1 and probe 2 were rationally designed and synthesized by hybridizing IR780 emitted in the first near-infrared regions(NIR-I,700-900 nm)and reported Aβ species sensing fluorescence probe CRANAD-58 through the strategy of substitution with privileged structure.As compared with probe 1 and CRANAD-58,probe 2 exhibited more red-shifted emission wavelength both in CH2Cl2 and in the state of binding with Aβ species,which is due to stronger electron-donating ability of probe 2.In addition,probe 2 also showed a fast binding kinetics process with Aβ species and high sensitivity and selectivity towards Aβ species.In vivo NIR fluorescence imaging showed that probe 2 is able to distinguish 10-month-old APP/PS1 transgenic mice from wild-type mice.The histological staining of brain tissue displayed that probe 2 can quickly penetrate the blood-brain barrier(BBB),and stain both the core Aβ plaque and the Aβ oligomer resided on the peripheral areas of plaques.Subsequently,on the basis of probe 2,further structural modification was conducted to obtain probe 3-9.Amongst these probes,probe 9 produced excellent in vivo performance due to its higher brain uptake and improved pharmacokinetic properties in relation to probe 2,which was reflected by its increased discrimination between transgenic AD mice and control mice(～1.7 fold v.s.～1.3 fold).Probe 9 also possessed the capability to distinguish 6-month-old APP/PS 1 transgenic mice from wild type mice,suggesting its successful detection of Aβ species in the early stage of AD in vivo.Furthermore,the dynamic real-time detection of Aβ species in microregion of brain via upright fluorescence microscope indicated that probe 9 could quickly penetrate the blood-brain barrier,and then light up Aβ species in brain parenchyma and cerebral vessels(CAAs).In conclusion,probe 9 could specifically detect soluble and insoluble Aβspecies,and then effectively distinguish APP/PS1 transgenic mice from control wild-type mice,indicating that probe 9 could act as a useful tool for definite early diagnosis of AD and evaluation of the efficacy of treatment with anti-Aβ species drugs.In the second part,based on probe 2,a NIR fluorescence/PET bimodal probe(2-NODA[68Ga])was developed by introducing NOD A chelating agent into the structure and subsequent radio-labelling of 68Ga for sensitive imaging of Aβ species.The probe showed strong fluorescence response with and binding affinity to Aβ monomers,oligomers and aggregates,with the fluorescence emission wavelengths being 689 nm,683 nm and 689 nm,respectively,which fall in the optimal NIR region.The probe also displayed fast binding kinetics,high sensitivity and selectivity to Aβ species.In addition,the probe revealed good chemical stability and biocompatibility,and possessed a desirable logP value(logP=1.89).In vivo NIR fluorescent imaging verified that the probe is competent to discriminate 6-months-old or 22months-old APP/PS1 transgenic mice from its corresponding age-matched wild type mice,and showed that the difference between 22-months-old APP/PS1 transgenic mice and control mice is higher than that between 6-months-old transgenic mice and control mice.PET imaging displayed that the probe could quickly cross the blood-brain barrier with the initial brain uptake being～1.58%ID/g(4 min),and thus produced～1.57-fold PET signal in 9-months-old APP/PS1 transgenic mice than that of the control mice,suggesting that the bimodal probe could effectively discriminate transgenic mice from wild type mice.Additionally,the autoradiography studies showed specific signals of the probe with high density in brain slice of AD mouse,suggestive of its good penetrability of BBB and specific binding with endogenous Aβ species.In summary,the developed NIR fluorescence/PET bimodal imaging probe 2NODA-68Ga can successfully detect Aβ species in vivo through their respective characteristics of high sensitivity and high tissue penetration ability and importantly the complementary verification of each imaging result,so as to more efficiently realize the accurate diagnosis of AD.