Thiol-Reactive Star Polymers Functionalized with Short Ethoxy-Containing Moieties Exhibit Enhanced Uptake in Acute Lymphoblastic Leukemia Cells

Narges Bayat,1,2 Nathan McOrist,1 Nicholas Ariotti,3,4 May Lai,5 Keith CS Sia,1,2 Yuhuan Li,5 James L Grace,5 John F Quinn,5 Michael R Whittaker,5 Maria Kavallaris,2,6,7 Thomas P Davis,5,8,9,* Richard B Lock1,2,* 1Leukemia Biology Program, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia; 2School of Women’s and Children’s Health, Faculty of Medicine, University of New South Wales, Sydney, Australia; 3Electron Microscope Unit, Mark Wainwright Analytical Centre, Chemical Sciences Building, University of New South Wales, Sydney, NSW, Australia; 4School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia; 5ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, and Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia; 6Tumor Biology and Targeting Program, Children’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW, Australia; 7Australian Centre for Nanomedicine, ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of New South Wales, Sydney, NSW, Australia; 8Department of Chemistry, University of Warwick, Coventry, UK; 9Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD 4072, Australia*These authors contributed equally to this workCorrespondence: Richard B LockChildren’s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, PO Box 81, Randwick 2031, NSW, AustraliaTel +61 2 9385 2513Fax +61 2 9662 6583Email rlock@ccia.org.auPurpose: Directing nanoparticles to cancer cells without using antibodies is of great interest. Subtle changes to the surface chemistry of nanoparticles can significantly affect their biological fate, including their propensity to associate with different cell populations. For instance, nanoparticles functionalized with thiol-reactive groups can potentially enhance association with cells that over-express cell-surface thiol groups. The potential of such an approach for enhancing drug delivery for childhood acute lymphoblastic leukemia (ALL) cells has not been investigated. Herein, we investigate the impact of thiol-reactive star polymers on the cellular association and the mechanisms of uptake of the nanoparticles.Methods: We prepared fluorescently labeled star polymers functionalized with an mPEG brush corona and pyridyl disulfide to examine how reactivity to exofacial thiols impacts cellular association with ALL cells. We also studied how variations to the mPEG brush composition could potentially be used as a secondary method for controlling the extent of cell association. Specifically, we examined how the inclusion of shorter diethylene glycol brush moieties into the nanoparticle corona could be used to further influence cell association.Results: Star polymers incorporating both thiol-reactive and diethylene glycol brush moieties exhibited the highest cellular association, followed by those functionalized solely with thiol reactive groups compared to control nanoparticles in T and B pediatric ALL patient-derived xenografts harvested from the spleens and bone marrow of immunodeficient mice. Transfection of cells with an early endosomal marker and imaging with correlative light and electron microscopy confirmed cellular uptake. Endocytosis inhibitors revealed dynamin-dependent clathrin-mediated endocytosis as the main uptake pathway for all the star polymers.Conclusion: Thiol-reactive star polymers having an mPEG brush corona that includes a proportion of diethylene glycol brush moieties represent a potential strategy for improved leukemia cell delivery.Keywords: nanoparticles, targeting, endocytosis, leukemia, flow cytometry, microscopy