For biodistribution experiments, NCR nude female mice (Taconic) were used. To
attenuate gut fluorescence, an alfalfa-free special diet (AIN-93M Maintenance Purified
Diet from TestDiet) was administered to the mice 1 week prior to and during
experimentation. Nanoparticle formulations suspended in 1X PBS were administered
via the tail vein. In vivo imaging (IVIS, Caliper Instruments) was performed at
regular time points. Full scale biodistribution harvesting of relevant organs was
conducted at a relevant time point and imaged using the IVIS to quantify recovered
fluorescence. Circulation persistence experiments were performed in BALB/c female
mice from Taconic. Nanoparticle formulations were administered via the tail vein.
Blood collection was obtained from the retro-orbital sinus (w0.2e0.3 mL, diluted in
w0.1 mL 0.5 M EDTA) and imaged using the IVIS to quantify recovered fluorescence.
For cardiogreen recovery experiments, feces were recovered from three BALB/c mice
per experimental group and fractional recovered fluorescence (IVIS) was quantified
across a 48 h window. For co-injection experiments, free polymer corresponding to
the terminal layer polymer used for the NP architecture being investigated were
diluted in PBS and co-injected at normalized concentrations of particles against
10 mg/kg and 20 mg/kg free polymer in NCR mice. Experiments were performed
under the guidance and supervision of the MIT Department of Comparative Medicine
and Committee on Animal Care.