T rather of diffused drug derived from the nanoparticles, overall the data confirms that nanoparticles do coat the pegylated islets and thereafter release of cargo may continue over 3 weeks when cultured in vitro.2. Islet functionality is not impaired by encapsulationHaving established that islets can be decorated with a combination of PEG plus nanoparticles with preservation of islet structure, we next determined functional integrity of the encap-Figure 4. Prolonged viability of encapsulated islets in vitro. (A) Staining of viable (green) versus dead (red) cells in cultures of naked islets (CTR), pegylated islets (PEG), or pegylated plus empty-nanoparticle islets (Nano) at 1 d, 7 d, 14 d, and 21 d. (B) Percentages of viable cells in the different groups during culture. (C) Insulin staining in naked (CTR) and PEG-Nano-coated islets at 2 d and 14 d culture: more insulin positive cells were observed in islets encapsulated with nanoparticles (lower panels) compared to naked islets (upper panels). At least 10 islets were included in each group. Red BIBS39 custom synthesis represents insulin staining, blue staining (DAPI) represents nuclear staining in all cells. * p,0.05 and ** p,0.01. doi:10.1371/journal.pone.0050265.gNanotherapeutic Immuno-Isolation for Islet GraftsFigure 5. Prolonged functionality of encapsulated islets in vivo. Pancreatic islets from DBA/2 mice were grafted under the kidney capsule of C57BL/6 recipients: the islets were either untreated (Ctrl); or encapsulated in PEG alone (PEG alone); or with PEG decorated with empty nanoparticles (PEG+Empty Nano); or with PEG decorated with LIF-containing nanoparticles (PEG+LIF-Nano). The ability of these grafts 1317923 to support normoglycemia over 100 d is shown as “ survival” in (A). Histology of grafts taken from recipients showing normoglycemia at 100 d revealed well-preserved b cells containing insulin, as illustrated in (B). doi:10.1371/journal.pone.0050265.gsulated islets in terms of ability to MedChemExpress 58-49-1 respond to glucose stimulation. Comparing naked islets with encapsulated islets cultured 1 h in low glucose (2.8 mM) then with 1 h high glucose (28 mM), after overnight in primary culture, we found similar glucose response profiles for insulin release levels (Fig. 3A) with correspondingly similar stimulation indices (Fig. 3B). We deduced that the encapsulation process did not impair b cell function (i) in sensing glucose change and (ii) in responding to this change with insulin release.3. Prolonged viability of encapsulated islets in vitroIslet viability ex vivo is highly relevant to the potential use of harvested islets for clinical transplantation. We therefore compared naked versus encapsulated islets over a period of 21 d using low attachment conditions to mimic clinical harvest procedure. Three groups, naked islets, pegylated islets, and pegylated islets plus empty nanoparticles, were cultured in DMEM on low attachment cell culture plates. Live and died cells were analyzed at 1, 7, 14 and 21 days after culture using Syto Green and EB staining. Fig. 4A and B shows that, although viability at 1 d and 7 d was comparable across the three groups at around 75 , there was an unexpected prolongation of long-term viability at both 14 d (,72 ) and 21 d (,40 ) specifically associated with the combined PEG plus nanoparticles. This beneficial effect was significantly greater than pegylation alone 23727046 at 21 d. The pegylated islets without nanoparticles also showed marked benefits in terms of survival at 14 d (,60 ) and 21 d (,2.T rather of diffused drug derived from the nanoparticles, overall the data confirms that nanoparticles do coat the pegylated islets and thereafter release of cargo may continue over 3 weeks when cultured in vitro.2. Islet functionality is not impaired by encapsulationHaving established that islets can be decorated with a combination of PEG plus nanoparticles with preservation of islet structure, we next determined functional integrity of the encap-Figure 4. Prolonged viability of encapsulated islets in vitro. (A) Staining of viable (green) versus dead (red) cells in cultures of naked islets (CTR), pegylated islets (PEG), or pegylated plus empty-nanoparticle islets (Nano) at 1 d, 7 d, 14 d, and 21 d. (B) Percentages of viable cells in the different groups during culture. (C) Insulin staining in naked (CTR) and PEG-Nano-coated islets at 2 d and 14 d culture: more insulin positive cells were observed in islets encapsulated with nanoparticles (lower panels) compared to naked islets (upper panels). At least 10 islets were included in each group. Red represents insulin staining, blue staining (DAPI) represents nuclear staining in all cells. * p,0.05 and ** p,0.01. doi:10.1371/journal.pone.0050265.gNanotherapeutic Immuno-Isolation for Islet GraftsFigure 5. Prolonged functionality of encapsulated islets in vivo. Pancreatic islets from DBA/2 mice were grafted under the kidney capsule of C57BL/6 recipients: the islets were either untreated (Ctrl); or encapsulated in PEG alone (PEG alone); or with PEG decorated with empty nanoparticles (PEG+Empty Nano); or with PEG decorated with LIF-containing nanoparticles (PEG+LIF-Nano). The ability of these grafts 1317923 to support normoglycemia over 100 d is shown as “ survival” in (A). Histology of grafts taken from recipients showing normoglycemia at 100 d revealed well-preserved b cells containing insulin, as illustrated in (B). doi:10.1371/journal.pone.0050265.gsulated islets in terms of ability to respond to glucose stimulation. Comparing naked islets with encapsulated islets cultured 1 h in low glucose (2.8 mM) then with 1 h high glucose (28 mM), after overnight in primary culture, we found similar glucose response profiles for insulin release levels (Fig. 3A) with correspondingly similar stimulation indices (Fig. 3B). We deduced that the encapsulation process did not impair b cell function (i) in sensing glucose change and (ii) in responding to this change with insulin release.3. Prolonged viability of encapsulated islets in vitroIslet viability ex vivo is highly relevant to the potential use of harvested islets for clinical transplantation. We therefore compared naked versus encapsulated islets over a period of 21 d using low attachment conditions to mimic clinical harvest procedure. Three groups, naked islets, pegylated islets, and pegylated islets plus empty nanoparticles, were cultured in DMEM on low attachment cell culture plates. Live and died cells were analyzed at 1, 7, 14 and 21 days after culture using Syto Green and EB staining. Fig. 4A and B shows that, although viability at 1 d and 7 d was comparable across the three groups at around 75 , there was an unexpected prolongation of long-term viability at both 14 d (,72 ) and 21 d (,40 ) specifically associated with the combined PEG plus nanoparticles. This beneficial effect was significantly greater than pegylation alone 23727046 at 21 d. The pegylated islets without nanoparticles also showed marked benefits in terms of survival at 14 d (,60 ) and 21 d (,2.