Nanoparticle Functionalization with AMD3100

ANCs and ReANCs were functionalized with AMD3100 as previously described (Zevon et al., 2015 (link); Kantamneni et al., 2017 (link)). Briefly, AMD3100 (EMD Millipore, Burlington, MA) was dissolved in sterile water at a concentration of 1.25 μM and adsorbed onto nanoparticles by adding 388 μL of the AMD3100 solution to 3.5 mL of ANCs or ReANCs (1x concentration, i.e., total albumin concentration of 5 mg/mL), followed by constant agitation at room temperature for 3–4 h. This concentration of AMD3100 was found to be the optimal loading condition based on previously described in vitro experiments (Zevon et al., 2015 (link)). For particles that were dual loaded with AMD3100 and Dox (Dox-fANCs or Dox-fReANCs), AMD3100 functionalization was performed prior to Dox loading.