Research On Preparation And Properties Of Multifunctional NIR-Ⅱ Biological Nanoprobes

Fluorescence imaging（FI）is considered to be an important clinical imaging because of its high resolution and sensitivity,and it can monitor the pathological process in vivo in real time noninvasively.Generally speaking,the quality of FI is mainly determined by the sensitivity of the imaging camera and the fluorescence intensity of the probe.Imaging camera sensitivity has been improved a lot with the development of the optical imaging techniques.The way to further improve the quality of fluorescence imaging,to a large extent depends on the performance of image probe.Therefore,it is significant to obtain fluorescent probes with high quantum yield,high fluorescence intensity,high penetration depth and luminescence stability.At present,the imaging depth of the traditional visible and other near infrared fluorescent probes is limited within the scope of the 2 mm due to the heavy scattering absorption,and autofluorescence of biological tissue.This can only use in the imaging of biological skin,unable to realize high quality images in deep organs,which limits its application in clinical detection.Near-infrared-Ⅱ（NIR-Ⅱ）region（1000-1700nm）fluorescence probes with a longer wavelength has a deeper imaging depth due to the smaller absorption and scattering of biological tissues.Among them,rare earth ions(such as Nd³⁺,Er³⁺,Tm³⁺)can effectively improve tissue penetration depth and imaging quality,as their emission band is just in the NIR-Ⅱ region because of the special structure of 4f electron layer.However,rare earth doped nanomaterials often face the problems of fluorescence quenching caused by surface defects,low quantum yield in the near infrared second region and instability dispersed in water.In addition,with the development of tumor diagnostic probes,integrated diagnosis and treatment probes have been recognized as a new kind of fluorescent imaging probe.Through appropriate design,the probe can own fluorescence imaging and photothermal therapy,photodynamic therapy or other tumor treatment at the same time,which can not only simplify the treatment process,but also reduce the pain that patients experienced during the treatment.In order to solve the problems of low quantum yield and low penetration depth of existing fluorescent probes,rare earth doped nanomaterials with near-infrared excitation and NIR-Ⅱ emission were designed and prepared in this work.Their biological toxicity,luminescence performance and imaging performance were systematically studied.The main research results are as follows:（1）Na Er_xYb_1-xF₄nanoparticles with cubic crystal phase was synthesized by solvothermal method,and Na Er_0.8Yb_0.2F₄nanoparticles with an average size of 40±3nm exhibit the best fluorescence emission when adjusting the molar ratio of Er³⁺/Yb³⁺ions.Under the excitation of 980 nm,the nanoparticles exhibit strong fluorescence at1550 nm and the absolute quantum yield is up to 40.49%,it is 2 times of the existing reported similar materials.In order to obtain a highly stable nano dispersion for biological imaging,the surface of the nanoparticles was modified with polydopamine（PDA）.The quantum yield of the modified nanoparticles decreased to 29.23%,which is much higher than that of organic molecular probes used commercially（ICG,7.6%）.The dispersions of nanoparticles modified by PDA showed high photostability,and the fluorescence intensity remained unchanged under the irradiation of 980 nm laser（80 m W/cm²）for 2 h.Under the irradiation of 808 nm laser（0.5 W/cm²）,the nano dispersion（20mg/m L）can increase from 25℃to 60℃within 2 min,the conversion efficiency was determined as 60%.The cytotoxicity of the nanoprobes was evaluated and the hemolysis rate was 3.57%,which is lower than the international standard of5%.At a penetration depth of 5 mm in vitro,it has a high imaging resolution（～0.5mm）.The fluorescence signal was observed in the abdomen（liver and lung）of mice after injection for 3 h,and the signal-to-noise ratio was 5.1.The photothermal effect was also observed in mice.The probe is a potential multifunctional fluorescent probe for NIR-Ⅱ fluorescence imaging and photothermal therapy,which is expected to be applied in the future clinical diagnosis.（2）The Tm³⁺/Yb³⁺co-doped fluoride nanoprobe Na₅Y₈Tm_xYb_1-xF₃₂was prepared by solvothermal method.The optimum atom ratio of Tm³⁺/Yb³⁺doping was1/10.The particle size distribution of the probe is wide,ranging from 20 nm to 60 nm.Under 980 nm excitation,the characteristic emission peak of Tm³⁺at 1760 nm was measured.The surface of the nanoprobe was modified by PDA to obtain stronger dispersion stability in aqueous solution.After irradiation with 980 nm laser for 1 h,the fluorescence intensity of the nanoprobe did not change significantly,indicating that the nanoprobe has good photobleaching resistance.The absolute quantum yields of the nanoprobes before and after modification was 20.26%and 16.48%,respectively.The hemolysis rate of the probe was 2.84%,which met the international safety standard（<5%）and it showed that the nanoparticles exihibit low cytotoxicity.The probe has a longer emission wavelength than Er/Yb co-doped probe,which display smaller scattering and absorption loss.The photothermal conversion efficiency is 48%,and the penetration depth in pork tissue was determined as 10 mm in vitro,which is twice that of Na Er_0.8Yb_0.2F₄nanoprobe.The signal-to-noise ratio（SNR）of NIR-Ⅱ fluorescence images for Na₅Y₈Tm_0.1Yb_0.9F₃₂nanoprobes in mice was 9.3 when tail vein injection after 3 h,which is twice that of Na Er_0.8Yb_0.2F₄nanoprobe.It is a photothermal multifunctional probe with high penetration depth and NIR-Ⅱ fluorescence imaging.