Schematic representation and physicochemical characterization of islet-targeting nanomaterials. (A) Carbodiimide chemistry was used to covalently conjugate islet targeting peptide (CHVLWSTRKC) to the amphiphilic PLGA-b-PEG-COOH block co-polymer, which undergoes spontaneous self-assembly in aqueous solutions to form nanoparticles (B) ¹H-NMR spectrum of polymer-peptide conjugate displays the peaks characteristic of tryptophan (W) residue in the peptide (arrow), which is absent in the unmodified polymer. (C) Transmission electron microscopy (TEM) and dynamic light scattering analysis reveal that the islet-targeting nanoparticles have an average diameter of 190±40 nm. Scale bar on TEM image = 200 nm

Nanoparticle binding to islet capillary endothelial (CE) cells in static culture. (A) Under static culture conditions, nanoparticles displaying islet-targeting peptide (CHVLWSTRKC; Pep I) exhibit a 3-fold increased binding (***p<0.001) to islet CE cells compared to skin CE cells, as shown in the representative fluorescent images of coumarin-loaded nanoparticles and quantified in the bar graph. This preferential binding to islet CE cells is specific to Pep I since the scrambled peptide (PepX) containing the same amino acids does not exhibit any binding preference. Scale bar = 20 μm (B) Islet CE cells treated with coumarin-loaded nanoparticles were stained with lysotracker ™ red to label acidic organelles. Quantitative analysis of green (nanoparticle) and red (lysotracker) images reveals approx. 100% colocalization; Mx (green colocalizing with red) = 97.86%; My (Red colocalizing with green) = 99.99%

Nanoparticle binding to islet capillary endothelial (CE) cells under flow. To mimic nanoparticle binding to islet endothelium in vivo, islet and skin CE cells were cultured in parallel microfluidic channels and the NP-Pep I and NP-Pep X suspensions (10 ug/ml) were flown over them at a wall shear rate of 2 dyne/cm². Quantitative analyses of fluorescence images showed NP-Pep I binding specificity similar to that seen under static conditions (***p<0.001). Scale bar = 100 μm

Immunosuppressive effect of islet-targeting nanomaterials. (A) Genistein, a pharmacologic inhibitor of receptor tyrosine kinase, blocks leukocyte binding to TNF-α-stimulated islet CE cells in a dose dependent manner (***p<0.001). (B) Genistein was encapsulated into NP-Pep I nanoparticles at 5% (w/w) and drug release measured over 48 hours. (C) Islet CE cells treated with genistein-loaded islet-targeting nanoparticles (NP-Gen; 10 ug/ml) 18h prior to TNF-α stimulation exhibit significant inhibition in leukocyte binding (***p<0.001)

Regulation of therapeutic and physicochemical properties of NP-Gen as a function of drug and nanoparticle dosage. (A) NP-Gen concentration of 10 μg/ml produced the most significant inhibition (***p<0.001) in leukocyte/islet CE cell binding while higher doses were progressively less effective. This trend is attributed to the pro-inflammatory effect of nanoparticles, as indicated by increased leukocyte binding to islet CE cells treated with higher doses of blank NP-Pep I nanoparticles. (B) Increasing genistein loading into NP-Gen nanoparticles results in a significantly greater immunosuppressive effect, as indicated by a marked decrease in leukocyte binding to islet CE cells. (C) NP-Gen nanoparticles loaded with increasing amounts of genistein exhibit a progressive increase in their average size.