The Synthesis Of Near-infrared ? Excretable Rare Earth Doped Nanoparticle And Multifunctional Biomedical Imaging And Surgical Navigation

Part 1Objective:Benefiting from deep tissue penetration and high signal-to-noise ratio,fluorescence imaging in the second near-infrared window(NIR-?,1000-1700 nm)has attracted immense attention and holds great promise to visualize and monitor real-time physiological and pathological processes in vivo.Nevertheless,most of reported NIR-? nanoparticles are still suffering from their long retention in reticuloendothelial system(RES)and inability to clear from living body,which raise the potential safety concerns and greatly hamper their future broad biomedical applications and translation.We have designed and synthesized novel rear earth doped nanoparticles(RENPs)consisting of b-phase Na YF₄:Nd 7%@Na YF₄ and further coated with liposome to generate the excretable NIR-? nanoprobe,RENPs@Lips.Notably,over 90%of RENPs@Lips were excreted from the body within only 72 h after intravenous administration.In addition,it demonstrated the capability of multifunctional biomedical imaging in circulatory system associated disorders and surgical navigation.The purpose of the first part in this project was to synthesize the NIR-? nanoprobe RENPs@Lips and to evaluate its relevant characterizations.Methods:RENPs consisting of b-phase Na YF₄:Nd 7%@Na YF₄were synthesized using the thermal decomposition method in high boiling point organic solvent(1-octadecene and oleic acid)and further demonstrated by the transmission electron microscopy(TEM)and X-ray diffractiong(XRD).To improve the dispersion of RENPs in water,we mixed 1,2-dipalmitoyl phosphatidylcholine,cholesterol,and PEGylated lipid to synthesize the liposome and then to use the liposome to further coat the RENPs.The resulted nanoprobe RENPs@Lips displayed excellent water solubility.The relevant characterizations of the RENPs@Lips were further evaluated as follows:homogeneous size and dispersion by TEM,and element mapping analysis by TEM-EDS,absorbance in a UV-Vis/NIR spectrum,fluorescence emission and quantum yield under 808 nm excitation laser,hydrodynamic size and physicochemical stability in different media(PBS,DMEM/FBS,and plasma)by dynamic light scattering(DLS)and the optical stability in different media under 808 nm excitation laser.Results:The TEM result of RENPs@Lips demonstrated monodisperse and homogeneous size 72.2±1.6 nm(coating thickness:4.3±0.8 nm),and the P and O elements mapping demonstrated that liposome was coated on the RENPs successfully.Multiple absorbance peaks including one at 800 nm were observed dual emission peaks at 1064 nm and 1345 nm under 808 nm excitation with large Stokes shifts(264 nm and545 nm,respectively).The quantum yield of RENPs@Lips was calculated to be 7.9%at 1064 nm and 4.1%at 1345 nm under 808 nm excitation(IR26 as a reference 0.5%in1,2-dichloroethane).the mean hydrodynamic size of the nanoparticle was 105±5.2nm in PBS and remained the same over long time(25 h)without aggregation in different media(PBS,DMEM/FBS and plasma).The Zeta potential of the RENPs@Lips both in DMEM/FBS and in plasma were higher than that in PBS.In addition,the NIR-? fluorescent emission intensity of the RENPs@Lips didn't decrease in different media for 25 h under 808 nm excitation.Conclusions:The NIR-? nanoprobe RENPs@Lips with liposome coating demonstrated high quantum yield.They would not aggregate in water and blood,highlighting their outstanding optical performance and excellent physicochemical stability as well as photostability.Part 2Objective:The assessment of in vivo pharmacokinetics and biocompatibility of nanoprobe RENPs@Lips.Methods:To investigate in vivo pharmacokinetics,RENPs@Lips(1.0 mg/m L,200?L)were injected into nude mice via tail vein and monitored under NIR-? imaging setup.The mice were sacrificed at different time points post-injection,then vital organs and blood,feces and urines were collected.All the samples were imaged ex vivo under NIR-? setup and examined by inductively coupled plasma mass spectrometry(ICP-MS)level to analyze relevant half-lives.To evaluate the cytotoxicity of RENPs@Lips,a standard MTT analysis was performed on the embryonic fibroblast cell NIH 3T3 and microphage cell RAW 264.7.To examine in vivo toxicity,certain behaviors and weight changes were recorded during the entire observation period at different injection doses of RENPs@Lips.Further histological analyses and blood chemistry profiles were investigated at different time points post-injection.Results:NIR-? imaging of whole-body was performed and revealed that RENPs@Lips were mainly accumulated in the liver and spleen shortly post-injection.It is noteworthy that the fluorescent signals of liver and spleen exhibited a quick decline from 16 h to 28 h and very weak signals could be visible at 72 h post-injection.Notably,the fluorescent signal of intestines and feces sharply increased within 16 h and then declined over time and reached to very low level at 72 h post-injection.The fluorescence analyses showed that over 90%of RENPs@Lips were excreted from the liver within 72 h after intravenous administration and in vivo half-lives of RENPs@Lips in blood,liver and spleen were calculated as 17.96 min,23.0 h and 14.9 h,respectively.Meanwhile,the ex vivo fluorescence analysis and quantitive ICP-MS level of vital organs,feces and urines showed high consistence with our in vivo NIR-? imaging data.It was observed that RENPs@Lips with different doses exhibited no apparent cytotoxicity to both NIH 3T3 and RAW 264.7 cells.Compared to the control groups,all the mice exhibited no signs of abnormal behavior of toxicity and no significant difference in weight changes during the entire observation period of 30 days at different injection doses(1.0 mg/m L and 2.0 mg/m L).In addition,the vital organs(heart,liver,spleen,lung and kidneys)of the mice were harvested and demonstrated no obvious hydropic damage or necrotic lesions at 1-and 7-days post-injection with dose of 1.0mg/m L.Meanwhile,blood chemistry profiles also revealed no significant alteration compared with the control groups.Conclusions:Over 90%of RENPs@Lips could be excreted out of body within 72 h mainly through hepatobiliary route,indicating that the most difficult challenge in metabolic concerns of current nanoprobes could be addressed.These outstanding pharmacokinetics and biocompatibility demonstrated RENPs@Lips were appropriate for further in vivo applications in identifying and monitoring diseases and surgical navigation.Part 3Objective:The assessment of multifunctional biomedical imaging and surgical navigation of RENPs@Lips in circulatory system associated physiological and pathological processes in vivo.Methods:For the blood circulatory system,femoral arterial thrombus murine model was induced by 5%Fe Cl₃,acute ischemia murine model was established by ligation of the femoral artery,and osteosarcoma-bearing murine model was constructed by subcutaneous injection of 143B cells,respectively.The in vivo diagnostic imaging was recorded under NIR-? setup after injection of RENPs@Lips via tail vein.Furthermore,the femur orthotopic osteosarcoma mice model was established and the separation and ligation for tumor blood supply was performed according to the NIR-? imaging navigation.For the lymphatic circulatory system,to visualize and resect the sentinel lymph nodes,RENPs@Lips were injected intradermally near the base of the tail and recorded under NIR-? setup.In addition,xenograft B16F10 melanoma murine model was generated and RENPs@Lips were injected intradermally at the margin of the melanoma.To precisely confirm metastasis range of tumor,the localization of sentinel and secondary lymph nodes were performed according to the lymphatic drainage tracking.Finally,the dissection was performed under NIR-? surgical navigation and analyzed by histological biopsy results to examine whether metastasis exists.Results:For the blood circulatory system related diseases,the favorable and precise delineation of hemodynamics in artery thrombosis,acute ischemia and tumor angiogenesis were successfully accomplished under NIR-? imaging after the intravenous injection of RENPs@Lips.Moreover,RENPs@Lips displayed feasibility of real-time monitoring the reconstruction of collateral vessels in blood perfusion in response to acute ischemia.Meanwhile,the major blood supply of orthotopic osteosarcoma was accurately separated and ligated to complete the NIR-? image-navigated embolization surgery in vivo.For the lymphatic circulatory system,at 6 h post-injection of RENPs@Lips at the base of the tail,the inguinal lymph node was clearly visualized and precisely resected with bare normal tissue remained under NIR-? imaging navigation.Furthermore,after RENPs@Lips were injected intradermally at the margin of the melanoma,the sentinel lymph node and secondary lymph node successfully located with the help of NIR-? tracking in lymphatic drainage.Under the NIR-? imaging navigation,the sentinel lymph node was firstly dissected with a positive(+)histological analysis.The secondary lymph node was subsequently dissected and histological analysis demonstrated negative(-),implicating the accomplishment of sentinel lymph node biopsy.Conclusions:NIR-? nanoprobe RENPs@Lips displayed favorable capatibility in timely identifying and dynamically monitoring the circulatory system associated disorders,providing a non-invasive and real-time alternative in pre-clinical trials of drug investigations.Meanwhile,the good performance of RENPs@Lips in NIR-? surgical navigation made it a potential platform for future applications in clinical tumor surgery.