Fig. 4

Optimized NCs do not cause toxicity during RH. a Mouse and human RBCs were mixed either with an agglutinating cross-species serum (dog serum) or NCs and then prepared as a “thin smear” slide. Dog serum causes RBCs to aggregate, as do bare PS-NPs, but nanogels do not. Scale bar represents 30 μm. b Round-bottom well assay of RBC aggregation, in which aggregated RBCs form a diffuse haze, while non-aggregated RBCs settle into a tight red dot. c RH nanogels, with the RBCs labeled with Cr-51, were prepared as in Fig. 2 and injected into the mice, followed by sacrifice after 30 min and organ Cr-51 signal measurement on a gamma counter. As a positive control, RBCs were intentionally aggregated with the anti-RBC antibody, Ter119, plus a cross-linking secondary antibody. Each data point represents mean ± s.e.m (n = 3). d, e RH nanogels and Ter119-aggregated RBCs were prepared as in c, but before sacrifice, the pulmonary artery pressure (PAP) was measured. Five min after injection, Ter119-aggregated RBCs (positive control) had increased the PAP, while RH nanogels had not (quantification in e). Each data point represents mean ± s.e.m (n = 4). f, g Mice were injected as in c and then had their blood oxygen measured over time (f), followed by their sacrifice at 30 min for histology (g), showing no difference between RH and RBCs-only. Scale bar represents 100 μm. Each data point represents mean ± s.e.m (n = 4). h Mice were injected with RH or free nanogels and intratracheally instilled with LPS, followed by measurement of the bronchoalveolar lavage (BAL) levels of leukocytes and protein, both measures of lung inflammation and ARDS. Each data point represents mean ± s.e.m (n = 3). *P < 0.01, non-paired, two-tailed t-test