Synthesis And In Vivo Bioimaging Of Sub-10nm Core-based NIR Rare-earth Doped Optical Probes

Rare earth doped nanoparticles（NPs）constitute a class of inorganic optical nanomaterials whose fluorescence arises from the radiative transition of the inner 4f electrons.They have a set of superior optical properties,such as high photostability,absence of photobleaching and blinking,sharp emission bands,and long luminescence lifetimes,making them promising as optical probes for biomedical application.However,they suffer from the retention of NPs that raise concern on long time potential toxicity,the low fluorescence quantum yield,and tissue-depth-dependent luminescence intensity which prevents them for accurate in vivo sensing.To overcome these problems,in this thesis,hexagonal NPs with low phonon lattice energy were selected to prepare sub-10 nm NPs.We aim to develop a class of sub-10nm rare-earth doped NaLnF₄ NPs with hexagonal phase that enables them to be cleared efficiently from the body.Moreover,these sub-10 nm NPs were utilized to build a hierarchical core/multishell NPs with superior optical property for high resolution optical bioimaging and temporal multiplexed bioimaging.These works include the following sections:We developed a strategy for synthesizing sub-10 nm rare earth doped NaLnF₄（Ln=Y,Gd,Er,Yb,Lu）of with hexagonal phase.Studies reveal that a precise control of the reaction temperature,the reaction time,allows us to achieve nanocrystals of hexagonal phase-a low symmetry phase suitable for efficient upconversion luminescence.Importantly,it was found that a simultaneous increase the amounts of Na OA（without OH^-）precursor and NH₄F,enables to dramatically reduce NaYF₄:30%Yb³⁺/2%Er³⁺NPs from 12.8 nm to sub-protein size of 5.4 nm.This is because the increase of Na⁺and F^-content can produce the increased number of crystal seeds,favoring the synthesis of smaller size hexagonal NPs.Indeed,a ratio of Na⁺:Re³⁺:F^-=8:1:8,with increased Na and F content,enables to synthesize a series of sub-10nm NaLnF₄ NPs（Ln=Gd,Er,Y,Yb and Lu）.Moreover,optimizing the doped concentration of Er³⁺and shelling the sub-10 nm NPs result in a 425-fold upconversion enhancement as compared to the well-established NaYF₄:30%Yb³⁺/2%Er³⁺NPs,ascribed to the increased number of emission centers and the effective suppression of surface quenching effect due to shelling.Furthermore,we utilized the Na Er F₄ core NPs,synthesized above,to develop a class of clearable,ultrabright shortwave-infrared-emitting nanoprobes（Na Er F₄@NaYF₄ NPs）for high resolution dynamic in vivo imaging.The Na Er F₄ NPs（Er-NPs）NaYF₄ were selected to carry out further optimization,as they emit the strongest among the group of NaYF₄:x%Er@NaYF₄（x=2-100%）NPs,and the excitation of808 nm avoids overlapping with the absorption of water（abundant in the body）and produces negligible thermal damage.We also made a systematic investigation on the influence of the crystal phase（hexagonal（β）and cubic（α））,the lattice mismatch between core and shell,the shell thickness and the doped Ce³⁺on NIR luminescence of Na Er F₄ NPs.The results indicated that the hexagonal phase of low lattice symmetry,the low lattice mismatch between core and shell,and the proper shell thickness（≥3nm）favor the emission output.The brightest nanoparticles were determined to beβ-phase core@shell Na Er F₄@NaYF₄ nanoparticles（β-Er@Y NPs,13.4 nm）,which have the absolute quantum yield of 10%.Moreover,after transferring to aqueous phase,these brightest NPs,i.e.,PAA modifiedβ-Er@Y,allow a through-skull brain vessel imaging and dynamic vasculature imaging of the whole body at a high resolution of≤58μm.We also tracked the distribution of NPs in main organs（heart,liver,spleen,lung and kidney）by evaluating the quantitative content of NPs metal elements at a set of defined timepoints,as well as in collected urine and feces within a long period of 14 days.A near-complete clearance（clearance rate>90%）of injected NPs were observed,primarily 85%through the hepatic route and 15%from the renal clearance route.Lastly,utilizing the synthesized sub-10 nm NaYF₄ NPs as the inner core,we build up a core/shell/shell/shell nanoparticle of NaYF₄@NaYb F₄@NaYF₄:Yb,Tm@NaYF₄ NPs for temporal multiplexed upconversion in vivo imaging.This tetra-domain nanostructure enables to regulate energy migration and upconverting processes within confined nanoscopic domains in defined ways,thus yielding high quantum yield upconversion luminescence（maximum～6.1%,0.11 W/cm²）with precisely controlled lifetimes that span two orders of magnitude（from 78 to 2157μs）.Moreover,the measured lifetimes were shown to be independent of p H values of surrounding solutions and tissue penetration depth,and deliver low thermal output in tissues,promising their uses for biology.Importantly,intravenous and subcutaneous administration of aqueous form NPs into a Kunming mouse demonstrates high contrast lifetime-coloured imaging of them in liver and two abdomen subcutis.In addition,two nanoprobes with a lifetime difference of 148μs was able to be discerned in vivo,indicating a high temporal imaging resolution of at least 148μs.