Labeling Stem Cells With Near-infrared Fluorescent Heptamethine Cyanine Dye IR-780 Iodide For Noninvasive Optical Tracking

BackgroundStem cell transplantation has become a promising strategy in encouraging tissue regeneration, repair and replacement from injures and diseases. Cell imaging and tracking are crucial for understanding the fate and the mechanisms of stem cell-based therapy. However, without reliable tracers, identification, localization and distinguishion of the implanted stem cells from the host are difficult. Optical imaging is an easy and real-time tool with high sensitivity and non-irradiation exposure for in vivo monitoring of stem cells. This technology is based on ex vivo labeling of stem cells with suitable fluorescent agents, subsequent injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. Near-infrared (NIR) excitable fluorescent contrast agents hold great promise for non-invasive in vivo imaging due to low tissue autofluorescence and deep tissue penetration in this region of the spectrum. The aim of this study was to label stem cells with a unique NIR agent, IR-780 iodide, and study the potential applications of IR-780 iodide as a novel NIR tracer for non-invasive in vivo imaging of stem cells.MethodsIn the first part of this study, the photoproperties of IR-780 iodide were investigated systemically by Cary Eclipse Fluorometer and Kodak In-Vivo FX Professional Imaging System. The stability of IR-780 iodide in serum at 37℃was also investigated. We also presented a detailed analysis of the in vivo biodistribution and pharmacokinetics of IR-780 iodide after intravenous injection into the mouse. In addition, the preliminary in vivo acute toxicity of IR-780 iodide was also evaluated.In the second part, a series of investigations of IR-780 iodide for in vivo NIR imaging were carried out. Intrasurgery guides for sentinel lymph node resection in small mice and larger pigs were performed by subcutaneous injection of IR 780 iodide with the does of 0.5nmol and 10nmol respectively. The sentinel lymph node mapping by indocyanine green (ICG) and Quantum dots (QDs) in mice were also investigated as comparable controls. As a NIR contrast agent, the fluorescein angiography by IR-780 iodide was also applied in rabbits.In the third part, we describe the potential application of IR-780 iodide as a NIR agent for stem cells labeling and tracking. The multipotent stem cells derived from rat skin dermis (rDMCs) were cultured and incubated with IR-780 iodide with varying concentrations and different time periods to investigate the optimal conditions of IR-780 iodide for cell labeling. The labeling efficiency, cellular localization and its effects on cell viability and differentiation were extensively investigated. To compare the labeling capability of IR-780 iodide with different types of cells, we also labeled human bone marrow stromal cells (hBMSCs), rat bone marrow stromal cells (rBMSCs) and green fluorescent protein (GFP) transgenic mice derived bone marrow stromal cells (GFP-mBMSCs).In the last part,the in vivo distribution of labeled rDMCs after intravenous injection was traced by whole-body NIR imaging. To promote the pulmonary passage of transplanted stem cells, the sodium nitroprusside (SNP), a vasodilator agent, was injected before cell transplantation and also accessed by whole-body NIR imaging.Results1. IR-780 iodide is a lipophilic cation with peak absorption at 780-nm and molecular weight of 667 Da which can be detected conveniently by a NIR fluorescent detection system. The fluorescence emission intensity of IR-780 iodide was strongly influenced by solvent. IR-780 iodide has a higher fluorescence emission than classic cyanine dye ICG especially in serum. After incubation in serum for 1 hour, the total fluorescence emission of IR-780 iodide showed a slightly increase at first 20mins and then maintained at a stable level which indicated that IR-780 iodide was pretty stable in serum. After intravenous injection at a dose of 0.2mg/kg, most of IR-780 iodide was cleared from vital organs of the host mice after a couple of days. The result of acute systemic toxicity test suggested that the IR-780 iodide had no conspicuous acute toxicity in mouse under a dose of 2mg/kg.2. The in vivo sentinel lymph node mapping studies showed that when compared with ICG or QDs, IR-780 iodide has a significant photobrightening effect and a longer retention time (>3 hours) in the lymphatic system which allows to acquire much higher signal to noise ratios both in vitro and in vivo. In addition, this agent has a unique stability after fixation in tissues which raises the possibility of developing new and sensitive means of detecting lymph nodes in harvested surgical specimens. IR-780 iodide can also be a contrast agent which used in fluorescein angiography and has higher fluorescence intensity and longer blood half-life than traditional NIR contrast agent, ICG.3. The uptake and retention of IR-780 iodide was time-dependent and a labeling time of 15min and a concentration of 20μΜwere observed as the optimal condition for labeling of DMCs. IR-780 iodide was detected in the mitochondria of cells and had a higher labeling efficiency and fluorescence intensity than ICG and IR-786. IR-780 iodide labeling did not significantly affect cell proliferation, differentiation and migration capacity when compared with unlabelled controls.4. Following whole-body NIR imaging, the pulmonary passage of stem cells were non-invasively visualized in rats after systemic transplantation of IR-780 iodide labeled stem cells through intravenous delivery. With this NIR imaging method, we further confirmed that pretreatment with sodium nitroprusside (SNP), a vasodilator agent, significantly reduced the cell trapping in the lung and increased the cell passage through the lung capillaries(p<0.05).Conclusions1. This is the first report of use IR-780 iodide as a NIR agent in biomedical applications. This agent has unique photoproperies and stability in serum and can perform well under conditions of high fluence rate and prolonged exposure to bodily fluids at core body temperature.2. IR-780 iodide performs well in applications such as lymphatic imaging and fluorescein angiography. The technology using IR-780 iodide permits high sensitive and real-time image guidance throughout surgery procedure. In addition, it also assists the pathologist in tissue analysis.3. IR-780 iodide can be used as a NIR optical imaging agent to label and track stem cells both in vitro and in vivo. This noninvasive NIR fluorescence imaging technique using IR-780 iodide enables us to monitor the distribution and migration of stem cells in living animal model in real time. The excellent biocompatibility and unique optical properties of IR-780 iodide may provide a useful non-invasive imaging tool for stem cell-based therapies.