Fig. 3.

RH NCs are taken up by endothelial cells and leukocytes of target organ capillaries. a Mice were IV injected with RH rhodamine-conjugated nanogels (NGs), sacrificed 30 min later, and then the lungs were fixed and sectioned. The sections were stained with an endothelial marker (VE-cadherin, blue) and a leukocyte marker (NIMP, green). Rhodamine-NG fluorescence localized to the capillary endothelial cells with small amounts of nanogel signal colocalizing with sparse leukocytes (top row 10×, bottom row 40×). Thick white lines represent scale bars of 100 µm. b Mice were given intratracheal LPS to model ARDS prior to RH NCs injection as in a. The top left panel is a 10× image, while the top right is a 40× magnification plus digital zoom of the same region. As seen in the top left image, red nanogels are present in an overlapping distribution with both endothelial cells (blue) and leukocytes (green). The two bottom panels are 40× images of another region of the tissue, but displaying only two markers each for ease of viewing. Thick white lines represent scale bars of 100 µm. c Macrophages were plated in flow chambers and NGs (labeled red) were introduced, either free or adsorbed onto RBCs (labeled green). As seen in two separate experiments using RH NGs (top two panels), the RH NGs (red) localize to the center of the macrophages, and the RBCs (green) transiently localize on the periphery of the macrophages. In two other experiments using free NGs (bottom two panels), very little NG signal localizes with the macrophages. Scale bars represent 20 μm. In the rightmost panel, we have quantified the gain in macrophage-co-localized red fluorescence (corresponding to nanogels) during the course (10 min) of each of these experiments. Each data point represents mean ± s.e.m (n = 4 for free NG, n = 12 for RH NGs); *P < 0.05, non-paired, two-tailed t-test