| Background:Leukemia is a kind of hematopoietic malignant characterized with proliferation of hematopoietic stem cells,which is one of the most incident and fateful malignant tumor among the younger people.Although,our understandings of underlying biology of the disease and the advanced treatment strategies have greatly improved,the fact that leukemia disease-free survival rate is still low with unsolved leukemia relapse problems.Moreover,the conventional leukemia chemotherapy gained benefits for patients meanwhile with some severely adverse side-effects.Like bone marrow suppression,gastrointestinal reactions,liver and kidney damage and impaired immune function,etc.In addition,the current basic leukemia diagnosis are based on bone marrow or peripheral blood cell morphological and chemical analysis,including karyotype analysis,immunological detection flow cytometry fluorescence in situ hybridization and PCR.But it is still lack of a specific targeting way for monitoring leukemia cells in real-time,especially for monitoring the minimal residual disease.Given the current problems in the diagnosis and treatment of leukemia,it is urgent need to develop new targeting strategy to specifically recognize leukemia cells.Recently,tumor targeting strategy gradually become a new hotspot in the research of the leukemia treatment,has attracted extensive attention.The present leukemia targeting strategies mainly contents monoclonal antibody,gene mutations,epigenetic modifications and tumor related signaling pathways.However,on account of the complexity of the leukemia cell genome itself and malignant cell heterogeneity,these strategies are only target specific types of leukemia cells and its clinical application is limited.Recent researches reveal that as one of the basic characteristics of most solid tumors,microenvironment hypoxia regulate tumor cells genetic instability,cell metabolism,tumor drug-resistance,tumor angiogenesis and invasion and metastasis,which implies an important strategy for tumor targeting research.Current hypoxic targeting strategies are mainly regulating hypoxic cells and HIF-1α signaling pathway which is the major molecular signaling response to hypoxia.In addition,as the most important cellular organelles,mitochondria play central role both in cellular vital and lethal functions in diverse physiological and pathological conditions associated with cell survival and death.Previous reports have suggested leukemia cells contain greater mitochondrial mass,have higher rates of oxidative phosphorylation,and depend more on fatty acid oxidation for survival when compared with those in normal cells.Our previous studies have identified a couple of near-infrared(NIR)heptamethine indocyanine small molecule with preferential accumulation property in mitochondria of cancer cells,providing a new strategy and promising prospects for solid tumor targeting,imaging and therapy.However,none of them exhibit high fitness for detecting hematological malignancies cells with sensibility and accuracy.In order to further improve the tumor targeting ability in peripheral blood,we synthesis the glycine derivatives modified heptamethine cyanine fluorescent small molecule to targeting and treat cancer cells in the peripheral blood(leukemia cells and circulation tumor cells CTCs).The further mechanism studies will provide new targeting strategies,at the same time,provide a new reference for the further revealing of the leukemia biological characteristics and the significance of circulating tumor cells in cancer development.Methods and Results:1.Synthesis of a class of new kind of heptamethine cyanine fluorescent small molecule,NIR-27 and NIR-26 by adopting two hydrophilic glycine modification of IR-780 in N-alkyl side chain with the identification of molecular structure.At the same time,evaluated the optical properties water solubility of NIR-26 and NIR-27.The biological characteristics,including acute toxicity,internal distribution and metabolism of mice were further studies on rats.The main researches contented as follows:1.1 Synthesis of a class of new kind of heptamethine cyanine fluorescent small molecule,NIR-26 and NIR-27,by modified hydrophilic glycine at the N-alkyl side chain of IR-780.It is not only retain the near infrared fluorescent characteristic but also significantly improved the water solubility more than 20000 times when compared with IR-780,which shows introduction of amino acid structure could synthesis a new water soluble small molecule.1.2 The absorption and fluorescence spectra of NIR-27 were investigated in methanol,serum,and PBS.The absorption and emission peak of NIR-26 and NIR-27 in different media(methanol,serum,and PBS)lie in the NIR region(700–900 nm).In addition,the water solubility and fluorescent intensity of NIR-26 was greatly improved when compared with IR-780.1.3 The internal distribution and metabolism of NIR-26 and NIR-27 in normal mice was shown that this kind of fluorescent small molecules was rapidly distribute in the liver,kidney and other organs with the blood circulation after intravenous injection.Further studied revealed a strong fluorescent signal in feces after 24 h indicating the small molecules was excreted out of the body through the liver bile secretion.1.4 Through the acute toxicity experiment of NIR-26 and NIR-27 in the rat,we found that after 100 times of the mark dose injection,the SD rats general condition was good,and no obvious body-weight reduction,abnormal changes of blood,biochemical index was found.Observation of tissue slice in rats founded no obvious abnormal changes.2.The in vivo flow cytometery was used to study the leukemia targeting properties of the NIR-26 in vivo.The leukemia cells xenograft nude mouse model and human leukemia mice model were established to study the leukemia targeting characteristics of the NIR-26 in vivo.The main researches is as follows:2.1 The NIR fluorescence of NIR-26 was collocated with the GFP fluorescent tumor tissue in GFP-labeled HL-60 xenograft nude mouse model.The NIR fluorescence signals of the dissected organs further confirmed the cancer-targeting ability of NIR-26 in vivo,fluorescence of NIR and GFP can be observed to be co-localized in the area of the tumor.The tumor tissues frozen section observation with a laser confocal microscope also revealed the collocation of the NIR fluorescence with GFP fluorescence.On the other hand,the tumor targeting effect and the near-infrared imaging features of NIR-26 was also verified in the Jurkat and K562 cells xenograft nude mice model.2.2 The human leukemia mice model was established by tail vein injection of GFP labeled HL-60 cells in the C57BL/6 mice after total body irradiation with 60 Co gamma rays.The GFP-labeled HL-60 cells could exist in the circulation system of mice for a long time and the number of the cells fluctuated in a certain range when using the in vivo flow cytometry instrument for a real-time dynamic monitoring.Further important organs and peripheral blood observation fund that leukemia cells mainly existed in the bone marrow and certainly distribution in the peripheral blood.At the same time,certainly infiltrating of leukemia cells in spleen and liver was observed,which proved that this method could build the body dissemination of acute myeloid leukemia mice model.2.3 After tail vein injection of NIR-26,both the 488 nm channel and 785 nm channel of the in vivo flow cytometery were used to detect the fluorescence of GFP and NIR.We found that the NIR fluorescent and GFP fluorescent signal could overlap well after the injection of NIR-26 for 10 days,and double labeled cells reached to 89.74% of the total number of GFP labeled cells,which indicated that NIR-26 could obviously identify the HL-60 cells in the circulation system after injection for 10 days.In addition,the PBMCs smear,femur and spleen frozen section of mice were observed by the laser confocal microscope,and found that the NIR fluorescence could co-located with the GFP fluorescence which proved that the NIR-26 could target leukemia cells in the peripheral blood of mice and its near-infrared imaging features.3.Study the leukemia targeting properties of the heptamethine cyanine fluorescent small molecule NIR-26 in vitro,and preliminarily explored possible targeting mechanism.The main researches contented as follows:3.1 After incubated PBMCs and HL-60 cells with NIR-26 in different concentrations and time,the NIR fluorescence was detected and found that the fluorescent intensity of HL-60 was higher than PBMCs after incubated with 5μM of NIR-26 for 20 min,which was selected as the optimum cells labeling condition of NIR-26 in vitro.In addition,another two kinds of leukemia cell lines K562 and Jurkat were labeled in this condition and also found the significant difference of the fluorescence signal intensity in Jurkat and K562 cell when compared with PBMCs.3.2 The GFP labeled HL-60 cells and normal PBMCs were mixed with 1:1 to simulate the peripheral blood of the leukemia patients in clinic.Then,the mixed cells were incubated with 5μM of NIR-26 for 20 min and found that NIR-26 can specifically targeted the GFP labeled HL-60 cells in the mixed cells,which indicated that NIR-26 could specifically targeting the leukemia cells in the peripheral blood.3.3 In the mechanism studies,we found that NIR-26 transport into leukemia cells in an active way and NIR-26 transport into leukemia cells was significantly inhibit after oxidative phosphorylation and glycolysis were inhibited respectively.3.4 After using the hypoxia incubator and OATPs transporters inhibitor BSP incubated leukemia cells respectively,we found that hypoxia conditions can promote NIR-26 into leukemia cells,and BSP could significantly inhibited the NIR-26 into leukemia cells.Moreover,the cells HIF-1α expression were raised by incubating with the low oxygen dose of cobalt chloride(CoCl2)and expression of the HIF-1α was inhibit by siRNA,and the entrance of NIR-26 in the cells and relative OATPs genes were detected,we found that hypoxia regulate NIR-26 entrance into leukemia cells through the HIF-1α/OATPs signaling pathways.4.The tumor targeting properties and NIR imaging features of NIR-26 was studied,and the CTCs targeting properties of NIR-26 was further studied in the clinical advanced lung cancer patients.The mainly researches as follows:4.1 The NIR fluorescence of NIR-26 was collocated with the GFP fluorescent tumor tissue in GFP-labeled of human lung cancer cells A549 xenograft nude mouse model.Furthermore,the NIR fluorescence signals of the dissected organs further confirmed the cancer-targeting ability of NIR-26 in vivo,fluorescence of NIR and GFP can be observed to be co-localized in the area of the tumor.The tumor tissues frozen section observation with the confocal microscope also revealed the collocation of the NIR fluorescence with GFP fluorescence.In addition,the tumor targeting effect and the near-infrared imaging features of NIR-26 in the solid tumors was also verified in the MKN-45 and U251 cells xenograft nude mice model.4.2 The GFP labeled A549 cells and normal PBMCs were mixed with 1:1 to simulate the peripheral blood of the advanced lung cancer patients in clinic.Then,the mixed cells were incubated with 5μM of NIR-26 for 20 min and found that NIR-26 can specifically targeted the GFP labeled A549 cells in the mixed cells,which indicated that NIR-26 could specifically target the PBMCs in the peripheral blood,and could be used to trace the CTCs.4.3 The PBMCs from the advanced lung cancer patients with bone metastases and healthy donor were incubated with 5μM of NIR-26 for 20 min respectively,and cell immunofluorescence staining methods was used to CTCs which expressed EpCAM.Under laser confocal microscope,NIR-26 could specifically target the GFP fluorescent CTCs in the PBMCs,and the NIR fluorescence could collocate with the GFP fluorescence,which indicated that NIR-26 could specifically target the CTCs in peripheral blood from advanced cancer patients.ConclusionOn the basis of the previous researches,we synthesized a class of new heptamethine dye with better water solubility and stability to target tumor cells in the peripheral blood,which was synthesis by embellishing with glycine structure.We confirmed that NIR-26 could selectively be enriched in the leukemia cells in vitro and vivo.It is of great significance for real-time dynamic supervision of tumor cells in the blood using the in vivo flow cytometry,for early diagnosis of leukemia,guiding the treatment and monitoring the prognosis.Meanwhile,NIR-26 can also be enriched in the circulating tumor cells(CTCs)in peripheral blood with metastatic solid tumors,which has a great prospect in disease surveillance and tumor metastasis in vivo.In addition,the mechanistic research demonstrated that HIF-1α/OATPs signaling pathway mediated NIR-26 be uptake into leukemia cells.Given hypoxia is the normal biological characteristics of tumor cells,it is of general significance to target heterogeneous tumor cells.Further research provides a new reference and basis for the targeting treatment of leukemia. |
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