MK and TPD are supported by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (project number CE140100036)

MK and TPD are supported by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (project number CE140100036). 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. test (MannCWhitney U) was applied to analyze the difference between the uptake of star polymers in B-ALL and T-ALL cells. The statistical analysis was performed using GraphPad Prism software (GraphPad, CA, USA). The results are presented as the mean standard error. A P value 0.05 was considered statistically significant. Results Synthesis and Characterization of Star Polymers Star polymers with varying coronal composition and thiol-reactive peripheral moieties were synthesized via an arm first methodology using RAFT polymerization. Two stars were synthesized incorporating a POEGA corona with either (i) thiol-reactive groups or (ii) non-reactive benzyl groups at the periphery (denoted as Star-OEGA-PDS and Star-OEGA-Bz, respectively (Physique 1). Benzyl-terminated linear POEGA (POEGA-BSPA) was prepared by polymerizing OEGA490 in toluene with BSPA, resulting in macromolecular chain transfer brokers with benzyl groups at the chain end distal from the thiocarbonylthio moiety (= 11,400 g/mol, = 1.22, Physique S1). Synthesis of Pyridyl disulfide-terminated POEGA (POEGA-PDS) was achieved by polymerizing OEGA490 in toluene with the chain transfer agent PDSD, yielding polymers with a thiol-reactive group at the periphery (= 10,200 g/mol, = 1.19, Figure S2). Open in a separate window Physique 1 Synthesis of star polymers. (ACC) Size exclusion chromatographs of star polymers. (A) POEGA stars with unreactive peripheral moieties (BSPA) (blue) and POEGA-BSPA arms (red). (B) POEGA stars with thiol reactive moieties (PDS) (blue) and POEGA-PDSD arms (red). (C) POEGA/PDEGA (50/50) stars with thiol reactive groups around the PDEGA arms (DEG), POEGA-BSPA arms (red) and PDEGA-PDSD arms (green). (D) Schematic of the star polymers. Abbreviations: Star-OEGA-Bz, Star polymers incorporating a POEGA corona with BSPA; POEGA, Poly oligo (ethylene glycol) methyl ether acrylate. These materials were then independently used to prepare core Rabbit polyclonal to PDK3 crosslinked star polymers (denoted as Star-OEGA-Bz and Star-OEGA-PDS) by chain extending with a difunctional crosslinking agent (= 62,100 g mol?1 and = 1.25; Physique 1B for A 922500 Star-OEGA-PDS = 69,100 g mol?1 and = 1.11). Importantly, the benzyl groups were preserved during the synthesis of the Star-OEGA-Bz star, with the peaks at 7.2C7.3 ppm clearly evident in the 1H NMR spectrum of the final purified material (Determine S3). Likewise, the pyridyl disulfide groups were also unaffected by the polymerization process, with the characteristic pattern of peaks at 7.25, 7.85 and 8.5 ppm clearly evident in the spectrum of the purified Star-OEGA-PDS (Determine S4). Analysis by DLS revealed the number average hydrodynamic A 922500 diameter to be 9 and 8 nm for Star-OEGA-Bz and Star-OEGA-PDS particles, respectively. Successful Cy5 labelling was confirmed by SEC with dual RI/UV/VIS detection, with the SEC trace detected at 646 nm overlapping with that detected by RI (Physique A 922500 S5). To examine how changes in the OEGA coating impact on cell association, a third star was prepared incorporating both OEGA and DEGA repeat units in the star corona (denoted as DEG). Homopolymers of DEGA are substantially more hydrophobic than homopolymers of OEGA, and typically form turbid solutions in water above 15C (i.e., they exhibit a so-called lower critical solubility temperature (LCST) of ca. 15C).28 Therefore, star polymers in which a proportion of the POEGA arms are substituted with PDEGA arms would be expected to.