Development Of Novel Near-infrared Fluorecent Fluorophore NIRCP-61 For Stem Cell Tracking And Cytoprotection

BackgroundCell transplantation has recently initiated increasing interests in regenerative medicine and numerous successes have been reported,but the clinical efficacy of exogenous stem cell therapies remains unsatisfactory because of the restricted cell survival and poor understanding of cell fate after transplantation which severely hampered translational research of cell-based therapy.Thus,development of multifunctional agents to address cytoprotective activity and cell tracking ability simultaneously will be very helpful in cytotherapy.To our knowledge,there are no such agents have been reported so far.Near-infrared(NIR)imaging possesses great potential for non-invasive in vivo imaging due to the properties of low tissue auto-fluorescence and deeper tissue penetration,which is currently available in biomedical imaging.Heptamethine cyanine dyes are representative NIR fluorescent agents and a couple of these dyes have been extensively used in cell labeling and tracking.Of them,the indocyanine green(ICG),which is approved for clinical use as diagnostic reagent for a few decades,is recently emerging as a potential NIR agent in clinic for cell labeling and tracking.However,most of organic dyes are membrane lipophilic dyes with extensive cell contamination and potential cytotoxicity,severely hurdle their potential in clinical application.In our recent work,we have identified a class of lipophilic NIR fluorescent heptamethine cyanine dyes,which are able to accumulate into cell mitochondria preferentially and possess unique optical properties for in vivo cell tracking and biomedical imaging.Our structure-activity relationship study and other reports have indicated that the heptamethine core with lipophilic cationic property is essential for mitochondrial targeting.Based on these foregoing studies,the development a new derivative of heptamethine dye with both cell tracking and cytoprotective activities will be very helpful in cell-based therapy.Methods1.The mitochondrial heptamethine dye(NIRCP-61)was designed and synthesized through the modification of N-alkyl side chains around heptamethine core.2.Four types of mesenchymal stem/stromal cells(MSCs)derived from different organs and species,including mouse bone marrow MSCs(mBMMSCs),rat dermis MSCs(rDMSCs),human umbilical cord MSCs(hUCMSCs)and human dermis MSCs(hDMSCs),were used to confirm the labeling capability of NIRCP-61.A series of assays were executed to inspect the cytotoxicity of NIRCP-61 on physiology of labeled cells and dye transfer to neighboring cells of NIRCP-61 labeled cells.3.In order to study the cytoprotection of NIRCP-61 agaist oxidative stress,hydrogen peroxide(H2O2)and ionizing radiation(IR)were used to mimic the cell damage caused by oxidative stress in vivo,intracellular ROS accumulation and cell DNA double-strand breaks(DSBs)were detected.Intracellular antioxidative defense ability of NIRCP-61 and the possible mechanisms were studied by Western Blot after cell preconditioning.4.Combined irradiation and wound injury rat models,partial body excessive irradiation exposure rat models and acute myocardial infarction rat models,were built and hUCMSCs preconditioned with or without NIRCP-61 were transplanted.Cell retention,cell survival and therapeutic effects of NIRCP-61-preconditioned hUCMSCs were detected to evaluate the in vivo cytoprotective activity of NIRCP-61 in multiple rat models.Results1.A novel mitochondrial heptamethine derivant(NIRCP-61)was developed to meets the requirements for cell tracking and cytoprotection simultaneously in cell therapy.2.The small-molecule fluorophore maintains the superior NIR fluorescent imaging properties and could be easily accumulated into cell mitochondria just by incubation without the application of additional transfection agents and/or procedures.As a cell labeling probe,NIRCP-61 has the advantages of excellent biocompatibility and fewer cell contamination to microenvironment compares to orther organic lipophilic membrane dyes.3.NIRCP-61 significantly alleviates the cell damage caused by acute oxidative stress and the cytoprotective effect of NIRCP-61 is achieved by inducing the intracellular cytoprotective molecules against oxidative stress via delicately modulating nuclear factor erythroid 2-related factor 2(Nrf2)and phosphoinositide 3-kinase/protein kinase B(PI3K/Akt)pathways.4.NIRCP-61 preconditioning significantly increases the retention and survival of hUCMSCs after transplantation,augments tissue repair and regeneration,and consequently improves the outcomes of cytotherapy in multiple animal models.ConclusionTo our knowledge,this is the first agent that simultaneously facilitates NIR cell tracking and cytoprotection.This novel mitochondrial heptamethine dye may represents an attractive therapeutic fluorophore for cell transplantation and offers a convenient approach to impel potential translation in clinical cell-based therapies.