title: CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia.
abstract: Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ∼ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ∼ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ∼ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ∼ 12 nm retained bright fluorescence over an extended duration of ∼ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ∼ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ∼ 8.2% in human peripheral blood cells (PBMCs) which are CD33(low). The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.
authors.affiliation: Amrita Centre for Nanoscience and Molecular Medicine, Amrita Institute of Medical Science, Cochin 682 041, India.
authors.forename: Archana
authors.initials: A
authors.lastname: Retnakumari
authors.forename: Jasusri
authors.initials: J
authors.lastname: Jayasimhan
authors.forename: Parwathy
authors.initials: P
authors.lastname: Chandran
authors.forename: Deepthy
authors.initials: D
authors.lastname: Menon
authors.forename: Shantikumar
authors.initials: S
authors.lastname: Nair
authors.forename: Ullas
authors.initials: U
authors.lastname: Mony
authors.forename: Manzoor
authors.initials: M
authors.lastname: Koyakutty
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology*; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D045744:Cell Line, Tumor; D002470:Cell Survival; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006046:Gold / Q000737:chemistry*; D006801:Humans; D016130:Immunophenotyping; D007249:Inflammation / Q000473:pathology; D015470:Leukemia, Myeloid, Acute / Q000473:pathology*; D008214:Lymphocytes / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry* / Q000633:toxicity / Q000648:ultrastructure; D011419:Propidium / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D063268:Sialic Acid Binding Ig-like Lectin 3; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D016207:Cytokines; D063268:Sialic Acid Binding Ig-like Lectin 3; D012710:Serum Albumin, Bovine; D011419:Propidium; D006046:Gold