One mL of USP saline was then added into the flask

One mL of USP saline was then added into the flask. degree of drug loading. In conclusion, CLL1-targeting nanomicelles have the potential to be used for targeted drug delivery to leukemia stem cells. Keywords:leukemia stem cells, nanoparticles, targeted therapy == INTRODUCTION == The concept of cancer stem cells has huge implications for the management of cancer [1,2]. Cancer stem cells have been identified in both hematological and solid malignancies, suggesting that presence of cancer stem cells may be a common feature of most malignancies. Malignancy stem cells can self-renew and regenerate more cancer cells. Therefore, in order to remedy cancer, malignancy stem cells must be eradicated. However, malignancy stem cells are chemoresistant compared to their progeny cancer cells [3,4]. The goal of this study is usually to demonstrate the feasibility of using drug-loaded nanoparticles that are designed to bind with high affinity and specificity to acute myeloid leukemia (AML) stem cells (LSC). Chemoresistance of LSC can be overcome with high-dose chemotherapy followed by bone marrow transplantation. However, high-dose chemotherapy is usually associated with severe toxicity and therapy-related mortality. Many patients are not eligible for this treatment because of co-morbidities. This is especially true for AML patients, who have a median age at diagnosis of 60 to 65 years. For bone marrow transplantation, autologous bone marrow or stem cells are usually not used because of contamination by LSC. Allogeneic hematopoietic stem cell transplantation is usually often associated with severe graft-versus-host disease, and is commonly not offered to elderly patients. Therefore, the development of chemotherapy that is specific for LSC is usually a critical unmet medical need. Among cancer stem cells, AML LSC have been best characterized, and many of their cell surface molecules are known. For example, the C-type lectin-like molecule-1 (CLL1) is known to be preferentially expressed on most AML LSC. Even though it is normally expressed on CD38+ myeloid progenitors, itis not on CD34+ CD38- hematopoietic stem cells [5,6]. Recently, two of our co-authors (Drs. Luo and Lam) developed a novel biocompatible nanomicelle drug delivery system comprised of a unique amphiphilic polymers called telodendrimers [7]. Telodendrimers consist of cholic acid, lysine and polyethylene glycol (PEG) covalently conjugated together, which imparts the ability to self-assemble into a water-soluble spheroid with a hydrophobic core capable of sequestering many types of drugs. Cholic acid, a primary component of bile acid, possesses a facial amphiphilic structure: a rigid steroid scaffold with four hydrophilic groups on one surface, and hydrophobic methyl groups on the other surface of the NK-252 scaffold. Lysine is usually a natural amino acid. PEG is usually inert and has been used to improve the pharmacokinetics of therapeutic drugs. This nanocarrier system has many attractive characteristics for drug delivery such as high drug loading capacity, narrow polydispersity, well-defined structure, easy chemical modification, physical and chemical stability, biocompatibility and biodegradability. In this project, we used the phage-display library method and discovered a series of peptides that NK-252 bind NK-252 specifically to CLL1. One of these ligands, CLL1-L1, was used to decorate the surface of our nanoplatform to form a novel targeting nanomicelle that we named LSC-targeting nanomicelle. Unlike solid tumors that primarily reside at the extravascular space and are accessible by nanotherapeutics mainly through enhanced permeability and retention effect, LSC and leukemic cells reside primarily inside blood vessels and bone marrow that are directly accessible by nanotherapeutics through NK-252 intravenous administration. We report herein that targeting nanomicelles displaying CLL1-L1 not only can attach the nanomicelles to the surface of cells expressing CLL1, but, more importantly, can deliver the nanomicelles and their content into the target cells. == MATERIAL AND METHODS == == Identification of ligands targeting CLL1 == The CLL1 cDNA expression vector was purchased from the American Type Culture Collection (Manassas, VA, USA) and was subcloned to a pcDNA3.1 expression vector. The C7C phage display peptide library was purchased from the New England Biolab (Ipswich, MA, USA). CLL1 was expressed in 5637 (bladder transitional cell carcinoma), A549 (non-small cell lung cancer) and HTB38 (colon cancer) cells for sequential panning to identify peptides that bind to CLL1. At the time when this project was performed, there was no commercially available anti-CLL1 antibody to detect the expression of CLL1. Therefore, we constructed CLL1-RFP (red fluorescence protein) chimeric gene, and cloned into the pcDNA3.1 expression vector in which RFP replaced part of the intracellular domain of CLL1 to allow the monitoring of Rabbit Polyclonal to EDG3 CLL1 expression. To eliminate any phage that may bind to the confounding cellsin vivo, the C7C library was subtracted with whole blood, peripheral blood mononuclear cells (PBMC), normal healthy hematopoietic stem cells left over from allogeneic stem cell transplantation, human umbilical vascular endothelial cells (HUVEC), fibroblasts, 5637, A549 and HTB38 cells, for 46 hours with each cell type, before each round of panning (SI, S1). The subtracted phage library was then sequentially panned against 5637, A549 and HTB38 cells.