Functional Modifications Of Serum Albumin For Theranostics Targeting Amyloid β-Protein Fibrillogenesis

Alzheimer’s disease(AD)is a neurodegenerative disorder.The aggregation and accumulation of amyloid β-protein(Aβ)in the brain is one of the pathological features of AD,so toxic Aβ aggregates have become important targets and biomarkers for the treatment and diagnosis of AD.In addition,oxidative damage caused by high levels of reactive oxygen species(ROS)is also one of the pathological events in AD.Hence,it is highly desired to develop multifunctional agents for efficient Aβ detection and inhibition as well as ROS scavenging.Bovine/human serum albumin(BSA/HSA)has been found to bind Aβ through its hydrophobic pocket and inhibit Aβ aggregation,but the inhibitory effect of BSA and HSA is weak and mono-functional,so further modifications are necessary to improve its performance.Inspired by the efficient inhibition mechanism of a natural basic protein,lysozyme,we firstly modified BSA with ethylenediamine to increase the density of positive charge distribution on the surface of BSA.Four basified BSA(BSAB)with different modification degrees(MDs)were prepared.The results showed that BSA-B with the highest MD,BSA-B4,exhibited efficient inhibition on Aβ aggregation at low concentrations,significantly superior over native BSA.Mechanistic studies revealed that such an improved inhibitory potency of BSA was greatly attributed to the enhanced electrostatic interactions with Aβ.The aminated BSA achieved a conversion of binding way to Aβ,leading to more diverse driving forces.BSA-B(especially BSAB4)could bind more Aβ monomers,oligomers,and protofibrils than BSA,thus powerfully suppressing the following Aβ fibrillization.The same results were obtained for the basified HSA(HSA-B).Then a theranostic agent,HSA-BFP,was subsequently fabricated by conjugating an Aβ-specific near-infrared(NIR)fluorescence probe(F)and by coupling a cellpenetrating peptide(Penetratin,Pen)onto HSA-B.A variety of analyses demonstrated that HSA-BFP could not only efficiently inhibit Aβ aggregation and cytotoxicity but also exhibit potent performance in the NIR fluorescence detection of Aβ fibrils with higher sensitivity and stability than free F.The experimental results with transgenic strain C.elegans indicated that administration of HSA-BFP could decrease amyloid accumulation and delay Aβ-triggered paralysis of AD worms.Moreover,HSA-BFP displayed an improved blood-brain barrier(BBB)permeability.Therefore,HSA-BFP is demonstrated to be a multifunctional theranostic agent targeting Aβ.Finally,HSA-BFP@CDs were synthesized by combining HSA-BFP with carbon dots(CDs)capable of scavenging ROS.Comprehensive in vitro and in vivo experimental studies documented that HSA-BFP@CDs maintained efficient NIR detection/imaging of Aβ deposits and inhibition of Aβ aggregation.More importantly,compared with HSA-BFP,HSA-BFP@CDs possessed enhanced antioxidant capacity to scavenge excessive ROS and alleviate oxidative damage caused by ROS,enabling the multi-targeted and multi-functional treatment.In this study,the functionalized modification of BSA and HSA achieved the integration of multiple functions such as effective imaging of Aβ plaques,inhibition of Aβ aggregation,penetration of BBB,and mitigation of oxidative stress,which provides new insights into the design and development of multifunctional agents targeting Aβfibrillogenesis.