Nanoworms were synthesized using a one-pot Molday and MacKenzie (52 (link)) precipitation method as described by us previously (35 (link)). The main variation of the protocol was the ratio of dextran and iron salts in the reaction as described in Figure 1 . The molar ratio between Fe2+ and Fe3+ was kept the same. After the synthesis, particles were dialyzed in double distilled water, filtered through a 0.45-μm filter (Millipore), and stored at 4°C. TEM imaging was conducted to visualize the iron oxide core using FEI Tecnai Spirit BioTwin electron microscope (Electron Microscopy Facility at the University of Colorado Boulder). Size and zeta potential measurements of NPs were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The intensity weighted size distribution peak value was used to report hydrodynamic diameters of NWs.
For dextran shell crosslinking with epichlorohydrin, a two-step procedure was used as described before (35 (link)). For modification of dextran hydroxyls, SPIO NWs prepared at low dextran/Fe ratio (3 g dextran per 133.4 mg Fe salts), or the corresponding crosslinked CL-NWs were washed by ultracentrifugation in anhydrous DMSO two times and resuspended in anhydrous DMSO at 5.0 mg/mL (Fe concentration) in a borosilicate glass vial in the presence of 1 mg/mL of 4-dimethylaminopyridine (DMAP). Then, 2 mg/mL of 2-(2-methoxyethoxy)acetyl chloride or 2 mg/mL of 2-methoxyethoxymethyl chloride were added to the nanoparticles under stirring. Nanoparticles were incubated under nitrogen atmosphere with stirring at 37°C overnight, washed 3× in DMSO, 2× in DDW by ultracentrifugation, and resuspended in PBS for complement measurement. For modification with acetic anhydride, chloroacetic acid, or chloroethanesulfonic acid, CL-NWs were resuspended in DDW at 5 mg/mL (Fe concentration), stirred for 30 min in 2N NaOH solution, and then reacted with acetic anhydride (5% v/v), chloroacetic acid (5 mg/mL), or chloroethanesulfonic acid (5 mg/mL) at 37°C overnight with stirring. The particles were washed by ultracentrifugation and resuspended in PBS.
For dextran shell crosslinking with epichlorohydrin, a two-step procedure was used as described before (35 (link)). For modification of dextran hydroxyls, SPIO NWs prepared at low dextran/Fe ratio (3 g dextran per 133.4 mg Fe salts), or the corresponding crosslinked CL-NWs were washed by ultracentrifugation in anhydrous DMSO two times and resuspended in anhydrous DMSO at 5.0 mg/mL (Fe concentration) in a borosilicate glass vial in the presence of 1 mg/mL of 4-dimethylaminopyridine (DMAP). Then, 2 mg/mL of 2-(2-methoxyethoxy)acetyl chloride or 2 mg/mL of 2-methoxyethoxymethyl chloride were added to the nanoparticles under stirring. Nanoparticles were incubated under nitrogen atmosphere with stirring at 37°C overnight, washed 3× in DMSO, 2× in DDW by ultracentrifugation, and resuspended in PBS for complement measurement. For modification with acetic anhydride, chloroacetic acid, or chloroethanesulfonic acid, CL-NWs were resuspended in DDW at 5 mg/mL (Fe concentration), stirred for 30 min in 2N NaOH solution, and then reacted with acetic anhydride (5% v/v), chloroacetic acid (5 mg/mL), or chloroethanesulfonic acid (5 mg/mL) at 37°C overnight with stirring. The particles were washed by ultracentrifugation and resuspended in PBS.
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