A novel peptide ligand directed targeted drug delivery to Myeloid-derived suppressor cells ( MDSCs ) in tumor bearing mouse

SUMMARY Cancer is one of the most malignant diseases affecting human health. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that were found to present during cancer development. They contribute to the negative regulation of immune responses during cancer. We would like to develop an effective drug delivery system specifically towards MDSCs. We will describe the identification and characterization of a novel peptide modified liposome formulation targeting the Myeloid-derived suppressor cells in vivo. INTRODUCTION Myeloid-derived suppressor cells (MDSCs) play a critical role in tumor associated immune suppression[1]. They are comprised of precursors of macrophages, granulocytes, dendritic cells and myeloid cells at earlier stages of differentiation. These cells are defined as Gr1CD11b cells in mice. They accumulate in large numbers in lymphoid tissues and tumor tissues of tumor-bearing mice[2]. These cells are now considered as one of the major factors responsible for tumor associated immune defects and are an attractive target for therapeutic intervention. The phage display technology allows expression of exogenous peptides on the surface of phage particles. It could provide a rapid means for the discovery of peptide ligands against specific targets[3]. In vivo application of phage display library screening has been successfully conducted to identify peptide ligands targeting various tissues at physiologically normal or diseased states[4]. There were examples using the in vivo screening method to search for tumor-homing peptides. In this study, a model of 4T1 cancer was established in BALB/c mice. We conducted a series of in vivo phage display library biopanning studies, introducing random phagepeptide library into rats via tail vein injection. We demonstrated that specific phage clones could bind to MDSCs and the binding was mediated by specific peptide sequences. We used the peptide ligands to construct active targeting liposome drug delivery systems. We would like to explore the feasibility of using such peptide ligand-directed liposome as targeted drug delivery systems for cancer. EXPERIMENTAL METHODS Phage display library screening experiment was done following the manual of the Ph.DTM-7 phage library kit. Briefly, mice bearing tumors were anesthetized and i.v. injected with a C7 phage library (New England Biolabs) in 200 μl Tris-buffered saline containing 4×10plaque forming unit (pfu) phage. After 30 min, mice were perfused through the heart using phosphate buffer saline. The MDSCs were isolated from tumor of tumor-bearing mice via magnetic selection using anti-CD11b microbeads and LS columns following the manufacturer’s instructions[5]. Then the cells were lysed and the phage was rescued by ER2738 host bacterial infection for 30 min at room temperature. The phage was amplified in bacterial liquid culture for 5 h and then was purified by PEG/NaCl precipitation for the subsequent rounds of selection. After 3 rounds of screening, the eluted phages were titered on LB/IPTG/Xgal plates and blue plaques were picked randomly for sequencing using the -96 gIII sequencing primer (5′-CCCTCA TAG TTA GCG TAA CG-3′). The peptide sequences selected by the phage library and palmitylated-peptide were synthesized by GL Biochem Co., Ltd (Shanghai, China) and confirmed by HPLC and MS analysis to be at least 95% pure. Liposomes were prepared using the thin film hydration and extrusion method containing HSPC:CHOL: DSPE-mPEG2000 : rhodamine DHPE at 2:1:0.1:0.02 molar ratios. The size distribution of the liposomes were routinely examined by dynamic light scattering (Zetasizer Nano ZS90, Malvern, UK). For the FACS study, MDSCs and Jurkat cells were seeded at 1×106 pure well in 24-Well plates.4T1 and H1299 cells were plated in 24Well plates until cells grown to 80% confluence. After 1 h incubation with various concentration fluorescein labeled peptides, cells were washed using PBS for 3 times and collected with cell scrapers. The cell associated fluorescence intensities were analyzed by a flow cytometer (BD FACSCalibur, Becton Dickinson, USA). RESULTS AND DISCUSSION The Ph.D.-7 phage display library was screened for binding to MDSCs. After 3 rounds, 10 blue phage plaques on the tittering plate were randomly selected for cloning and sequencing. The peptide sequences that mediated significant phage binding were identified and synthesized with fluorescence tagging. Figure 1. FACS studies of peptides binding to MDSCs(a),Jurkat (b),H1299(c) and 4T1(d) cells. 4 types of cells were incubated with fluorescein labeled peptides at a concentration of 1mM(A), 500μM(B), 50μM(C), 5μM(D), 0. 5μM(E), 0 μM (F), respectively for 1h at 37 °C. The fluorescence peptide bindings were analyzed using FACS. Various concentration of fluorescein labeled peptides were incubated with four types of cells. As seen in Figure 1, after peptide binding, the resulted MDSCs associated fluorescences were much higher than those without peptide binding . There is a significant concentration-dependent increase at micromolar concentrations. In comparison, the binding of peptide to other cell types including the Jurkat cell, H1299 and 4T1 cells were not concentration dependent. CONCLUSION We identified a specific peptide sequence that can bind to MDSCs. It may be used to direct targeted drug delivery to Myeloidderived suppressor cells. REFERENCES 1. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nature reviews Immunology. 2009;9:162-74. 2. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumor-bearing mice. Journal of immunology. 2008;181:5791-802. 3. Li J, Zhang Q, Pang Z, Wang Y, Liu Q, Guo L, et al. Identification of peptide sequences that target to the brain using in vivo phage display. Amino acids. 2012;42:2373-81. 4. Pande J, Szewczyk MM, Grover AK. Phage display: concept, innovations, applications and future. Biotechnology advances. 2010;28:849-58. 5. Zhang, Y. Hematopoietic CCL5 promotes tumor growth and metastasis via bone marrow generation of myeloid-derived suppressor cells. Cell Research. In press . ACKNOWLEDGMENTS We would like to thank Professor Xiaojing Ma for the generous gift of 4T1 cells. The work is being supported by NSFC 81274365.