S. Sigrist

In vitro uptake evaluation in Caco-2 cells and in vivo results in diabetic rats of insulin-loaded PLGA nanoparticles.

PLGA nanoparticles (NPs) are largely developed for biological applications but little is known about their uptake. Therefore, we focused our study on the modalities of insulin-loaded PLGA NPs transport across Caco-2 monolayers, and their hypoglycaemic effect on diabetic rats. Insulin-loaded PLGA NPs were formulated by a double emulsion solvent evaporation process. NPs mean diameter was between 130 and 180 nm. NPs were smooth and spherical with an entrapment efficiency above 80%. Fluorescently labeled NPs were incubated with Caco-2 cells to study the process of uptake and the intracellular fate by flow cytometry and confocal laser scanning microscopy. The kinetic of absorption was time-dependent and occurred by clathrin-mediated endocytosis. The intracellular traffic led to a basolateral exocytosis of NPs. In vitro studies and in vivo intraduodenal administration to diabetic rats showed that NPs were resistant in intestinal conditions long enough to allow both the intestinal absorption of NPs and the delivery of functional insulin in bloodstream. The resulting in vivo hypoglycaemic effect was similar to a long-acting insulin one. As no effect on glycaemia occurred after oral administration, further studies need to be conducted to protect NPs from the degradation occurring at the enteric level.

Perfluorocarbon emulsions prevent hypoxia of pancreatic β-cells.

As oxygen carriers, perfluorocarbon emulsions might be useful to decrease hypoxia of pancreatic islets before transplantation. However, their hydrophobicity prevents their homogenisation in culture medium. To increase the surface of contact between islets and Perfluorooctyl bromide (PFOB), and consequently oxygen delivery, we tested effect of a PFOB emulsion in culture medium on β-cell lines and rat pancreatic islets. RINm5F β-cell line or pancreatic rat islets were incubated for 3 days in the presence of PFOB emulsion in media (3.5% w/v). Preoxygenation of the medium was performed before culture. Cell viability was assessed by apoptotic markers (Bax and Bcl-2) and by staining (fluoresceine diacetate and propidium iodide). β-Cell functionality was determined by insulin release during a glucose stimulation test and. Hypoxia markers, HIF-1α and VEGF, were studied at days 1 and 3 using RT-PCR, Western blotting, and ELISA. PFOB emulsions preserved viability and functionality of RINm5F cells with a decrease of HIF-1α and VEGF expression. Islets viability was preserved during 3 days of culture. Secretion of VEGF was higher in untreated control (0.09 ± 0.041 μg VEGF/mg total protein) than in PFOB emulsion incubated islets (0.02 ± 0.19 μg VEGF/mg total protein, n = 4, p < 0.05) at day 1. At day 3, VEGF secretion was increased as compared to day 1 in control (0.23 ± 0.04 μg VEGF/mg total protein) but it was imbalance by the presence of PFOB emulsion (0.09 ± 0.03 μg VEGF/mg total protein, n = 5, p < 0.05). While insulin secretion was maintained in response to a glucose stimulation test until day 3 when islets were incubated in the presence of PFOB emulsion preoxygenated (0.81 ± 0.16 at day 1 vs. 0.75 ± 0.24 at day 3), the ability to secrete insulin in the presence of high glucose concentration was lost in islets controls (0.51 ± 0.18 at day 1 vs. 0.21 ± 0.13 at day 3). Atmospheric oxygen delivery by PFOB emulsion might be sufficient to decrease islets hypoxia. However, to improve islets functionality, overoxygenation is needed. Finally, maintenance of islet viability and functionality for several days after isolation could improve the outcome of islets transplantation.

Overexpression of Vascular Endothelial Growth Factor In Vitro Using Deferoxamine: A New Drug to Increase Islet Vascularization During Transplantation

During pancreatic islet transplantation, delayed and insufficient revascularization can deprive islets of oxygen and nutrients, resulting in cell death and early graft failure. Deferoxamine (DFO), an iron chelator, increases vascular endothelial growth factor (VEGF) expression in cells. The aim of this work was to study the effect of DFO on beta-cell and pancreatic islet viability as well as VEGF expression. beta-cell lines from rat insulinoma (Rin m5f) and primary cultures of pancreatic islets from Wistar rats were incubated with DFO (10, 100, and 1000 micromol/L). The viability was evaluated using fluorescein diacetate/propidium iodide for dying pancreatic islets and using cell titers for Rin m5f. Expression of VEGF messenger RNA (mRNA) was quantified using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, VEGF secretion was determined using enzyme-linked immunosorbent assays at 1 to 3 days after treatment. The addition of 10 micromol/L of DFO preserved Rin m5F viability at 24 hours after treatment (10 micromol/L; 101.33% +/- 5.66%; n = 7). However, 100 and 1000 micromol/L of DFO induced cell death (68.92% +/- 5.83% and 65.89% +/- 5.83%, respectively; n = 4). In the same way, viability of pancreatic islets in the presence of DFO was preserved. RT-PCR analysis showed stimulation of VEGF mRNA in the presence of 10 micromol/L of DFO in islets at 3 days after culture. Finally, 10 micromol/L of DFO stimulated secretion of VEGF 7.95 +/- 0.84 versus 1.80 +/- 1.10 pg/microg total protein with 10 micromol/L of DFO in rat islets at 3 days after culture, n = 3; P < .001). The use of DFO to stimulate VEGF expression and increase islet vascularization may be a realistic approach to improve islet viability during transplantation.

Prevention of adhesion and promotion of pseudoislets formation from a β-cell line by fluorocarbon emulsions

Transplantation of pancreatic islets from cadaveric human donors is a promising therapy for type 1 diabetes. By reducing hyper/hypoglycaemic events, this therapy can strongly improve the quality of life of patients. There have been substantial advances in the islet-transplantation protocol (the Edmonton Protocol) through an improved islet-isolation method and the use of steroid-free immunosuppressants. Nevertheless, several difficulties continue to limit this approach. These include the gradual loss of islet graft functionality, side effects related to the use of immunosuppressants and, last but not least, the small number of potential donors. An alternate approach to solving the problem of the shortage of human islets consists of the transplantation of b-cell lines or b-cell surrogates. For this purpose, sources of b cells, such as pig and mouse pancreas and human and mouse embryonic stem cells are being investigated. Established bcell lines, such as the mouse insulinoma-6 (MIN6) cells offer several advantages over isolated islet material. The native pancreatic islet tissue is composed of various cell types (a, b and d cells). Furthermore, the sensitivity to glucose of b cells from freshly isolated islets exhibits considerable variability. In contrast, the MIN6 cell line provides a homogenous b-cell population whose insulin secretion responds physiologically to glucose stimulation. Under classical culture conditions, MIN6 cells adhere to uncoated, negatively charged tissue culture plastic to form mono ACHTUNGTRENNUNGlayers, but when grown on collagen-derived substrates, they form islet-like structures (pseudoislets) after 6–8 days of culture. Due to a lack of cell-to-cell contact, b cells from mono ACHTUNGTRENNUNGlayers are considerably less responsive to glucose and other nutrients than b cells arranged as pseudoislets. Hence, pseudoislets offer a better experimental model for studies of b-cell functionality than monolayer cells. In this study, we report that a perfluorocarbon (PFC) emulsion is able to prevent the adhesion of MIN6 cells onto negatively charged tissue culture plastic. Furthermore, the cells were seen to rapidly aggregate into well-formed pseudoislets. Owing to the specific properties of PFCs mentioned below, this approach to pseudoislet formation has the advantage of being nonimmunogenic, in contrast to the collagen-derived substrate approach. Owing to exceptional biological inertness, excellent spreading properties, high oxygen solubility and extremely low solubility in water, PFCs are being investigated for various biomedical applications. These include, in particular, intravascular oxygen transport, diagnosis, lung-surfactant replacement and drug delivery. Within the framework of the present investigation, the oxygen-carrying capacity of PFCs is also expected to prevent the hypoxia that contributes to the failure of post-transplant survival. This study represents an initial step whose aim is to establish the effects of PFCs on the viability and morphology of the MIN6 cell line. A PFC emulsion stabilized with Pluronic F-68 has been shown to reduce the endothelial adherence of sickled red cells. In that study, however, the reduction of adherence was also obtained with a PFC-free aqueous solution of Pluronic F68. The result was, therefore, attributed to a lubricating effect of the surfactant. In the present study, two hydrocarbon (HC) oil emulsions (Intralipid@ and a soybean oil (SBO) emulsion) that use the same surfactant as the PFC emulsion, provided the appropriate controls for assessing the effect of the PFC. Other studies have shown a modification of cell adhesion by using either pure liquid PFCs (cell culture at a liquid/liquid interface) or PFC-functionalized solid substrates. In both studies, a tendency to reduction of cell adhesion and promotion of spherical cell shape (reduced cell spreading) was observed. In the liquid/liquid system, cell growth was in the form of island monolayers, rather than in a continuous sheet monolayer of cells. In the present study, a 70% w/v-concentrated perfluorooctyl bromide (C8F17Br) emulsion, emulsified with egg yolk phospholipids (EYP, 4.67% w/v) and stabilized by a semifluorinated alkane, C6F13C10H21 (F6H10, 3.27% w/v), was used. [27] This PFC emulsion has a small average droplet size (~80 nm average diameter after terminal heat sterilization) and a narrow particlesize distribution. The mean diameter of the PFC droplets was only 100 nm after five months of storage at 4 8C. This PFC emulsion as well as the HC emulsions (Intralipid and a 45% w/v SBO emulsion) used as controls were diluted with an equal volume of twice-concentrated culture medium (DMEM) and added to the culture wells (see Experimental Section). The MIN6 cells were then incubated in DMEM containing various percentages (v/v) of these 1:1 diluted emulsions. For clarity, the results are expressed in terms of % v/v of the 1:1 diluted emulsions in the culture medium as well as, in the case of the PFC emulsion, % w/v of PFC in the total DMEM+emulsion volume. The effect of the PFC and HC emulsions on MIN6 cell cultures was investigated by counting the number of cells that remained adhered to the substrate. This number was compared with that obtained for control experiments in which the cells [a] Dr. M. Sanchez Dominguez, Dr. M. P. Krafft Syst mes Organis s Fluor s Finalit s Th rapeutiques (SOFFT). Institut Charles Sadron (CNRS) 6 rue Boussingault. 67083 Strasbourg, Cedex (France) Fax: (+33)388-414-099 E-mail : krafft@ics.u-strasbg.fr [b] E. Maillard, Dr. S. Sigrist, Dr. A. Belcourt Centre Europ en d’Etude du Diab te (CeeD) 1 boulevard Ren Leriche 67200 Strasbourg (France) Fax: (+33)390-201-219 E-mail : severine.sigrist@wanadoo.fr

Design, characterisation, and bioefficiency of insulin-chitosan nanoparticles after stabilisation by freeze-drying or cross-linking.

Insulin delivery by oral route would be ideal, but has no effect, due to the harsh conditions of the gastrointestinal tract. Protection of insulin using encapsulation in self-assembled particles is a promising approach. However, the lack of stability of this kind of particles in biological environments induces a low bioavailability of encapsulated insulin after oral administration. The objective of this work was to evaluate the effect of two stabilisation strategies alone or combined, freeze-drying and cross-linking, on insulin-loaded chitosan NPs, and to determine their bioefficiency in vitro and in vivo. NPs were prepared by complex coacervation between insulin and chitosan, stabilised either by cross linking with sodium tripolyphosphate solution (TPP), by freeze-drying or both treatments. In vitro bioefficiency NP uptake was evaluated by flow cytometry on epithelial models (Caco-2/RevHT29MTX (mucus secreting cells)). In vivo, NPs were injected via catheter in the peritoneum or duodenum on insulinopenic rats. Freeze-drying increased in size and charge (+15% vs control 412 ± 7 nm; + 36 ± 0.3 mV) in comparison with cross linking which decreased NP size (-25%) without impacting the NP charge. When combined the consecutive treatments reduced NPs size and increased charges as compared to standard level. Freeze drying is necessary to prevent the destruction of NP in intestinal environment in comparison with no freeze dryed one where 60% of NP were destroyed after 2h. Additionally freeze drying combined with cross linking treatments improved bioefficiency of NP with uptake in cell increased when mucus is present. Combination of both treatment showed a protection of insulin in vivo, with a reduction of glycemia when NPs were administrated. This work showed that the combination of freeze drying and cross linking treatment is necessary to stabilize (freeze-drying) and increase bioefficiency (cross-linking) of self assembled NP in the delivery of insulin in vitro and in vivo.

Pro-Inflammatory and Pro-Oxidant Status of Pancreatic Islet In Vitro Is Controlled by TLR-4 and HO-1 Pathways

Since their isolation until implantation, pancreatic islets suffer a major stress leading to the activation of inflammatory reactions. The maintenance of controlled inflammation is essential to preserve survival and function of the graft. Identification and targeting of pathway(s) implicated in post-transplant detrimental inflammatory events, is mandatory to improve islet transplantation success. We sought to characterize the expression of the pro-inflammatory and pro-oxidant mediators during islet culture with a focus on Heme oxygenase (HO-1) and Toll-like receptors-4 signaling pathways. Rat pancreatic islets were isolated and pro-inflammatory and pro-oxidant status were evaluated after 0, 12, 24 and 48 hours of culture through TLR-4, HO-1 and cyclooxygenase-2 (COX-2) expression, CCL-2 and IL-6 secretion, ROS (Reactive Oxygen Species) production (Dihydroethidine staining, DHE) and macrophages migration. To identify the therapeutic target, TLR4 inhibition (CLI-095) and HO-1 activation (cobalt protoporphyrin,CoPP) was performed. Activation of NFκB signaling pathway was also investigated. After isolation and during culture, pancreatic islet exhibited a proinflammatory and prooxidant status (increase levels of TLR-4, COX-2, CCL-2, IL-6, and ROS). Activation of HO-1 or inhibition of TLR-4 decreased inflammatory status and oxidative stress of islets. Moreover, the overexpression of HO-1 induced NFκB phosphorylation while the inhibition of TLR-4 had no effect NFκB activation. Finally, inhibition of pro-inflammatory pathway induced a reduction of macrophages migration. These data demonstrated that the TLR-4 signaling pathway is implicated in early inflammatory events leading to a pro-inflammatory and pro-oxidant status of islets in vitro. Moreover, these results provide the mechanism whereby the benefits of HO-1 target in TLR-4 signaling pathway. HO-1 could be then an interesting target to protect islets before transplantation.

Extracellular matrix proteins involved in pseudoislets formation

Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.

Improvement of rat islet viability during transplantation: validation of pharmacological approach to induce VEGF overexpression.

Delayed and insufficient revascularization during islet transplantation deprives islets of oxygen and nutrients, resulting in graft failure. Vascular endothelial growth factor (VEGF) could play a critical role in islet revascularization. We aimed to develop pharmacological strategies for VEGF overexpression in pancreatic islets using the iron chelator deferoxamine (DFO), thus avoiding obstacles or safety risks associated with gene therapy. Rat pancreatic islets were infected in vivo using an adenovirus (ADE) encoding human VEGF gene (4.108 pfu/pancreas) or were incubated in the presence of DFO (10 μmol/L). In vitro viability, functionality, and the secretion of VEGF were evaluated in islets 1 and 3 days after treatment. Infected islets or islets incubated with DFO were transplanted into the liver of syngenic diabetic rats and the graft efficiency was estimated in vivo by measuring body weight, glycemia, C-peptide secretion, and animal survival over a period of 2 months. DFO induced transient VEGF overexpression over 3 days, whereas infection with ADE resulted in prolonged VEGF overexpression lasting 14 days; however, this was toxic and decreased islet viability and functionality. The in vivo study showed a decrease in rat deaths after the transplantation of islets treated with DFO or ADE compared with the sham and control group. ADE treatment improved body weight and C-peptide levels. Gene therapy and DFO improved metabolic control in diabetic rats after transplantation, but this effect was limited in the presence of DFO. The pharmacological approach is an interesting strategy for improving graft efficiency during transplantation, but this approach needs to be improved with drugs that are more specific.

Duodenum-specific drug delivery: In vivo assessment of a pharmaceutically developed enteric-coated capsule for a broad applicability in rat studies

Targeting new oral drug formulations in the intestine has a broad applicability in animal studies. Enteric-coated capsules are gastroresistant and specific drug delivery systems useful for the evaluation of new pharmaceutical formulations during pre-clinical validations in rats. The purpose of this study was to develop and validate in a large-scale, reliable, reproducible capsules, to offer a safe and standardized duodenum-specific delivery system adapted for studies in rats. The reproducibility of the coating method, the coating layer uniformity and thickness, the external capsules integrity and their enteric properties after in vitro dissolution in simulated gastric and intestinal media were already evaluated and validated. This study presents the in vivo tests of the gastroresistance and of the location of the disintegration. Micro-computerized tomography and a pharmacokinetic study of acetaminophen-filled capsules showed that the enteric-capsules were resistant in the stomach with no apparent leak of the capsules, and were disintegrated in the early duodenum 1-1.5h after oral administration. A positive impact on the bioavailability of acetaminophen in coated capsules was attested. In conclusion, this work, developed with a rigorous pharmaceutical technology, presents a tool adapted for duodenum-specific delivery of new formulations in rats.

Adenoviral infection or deferoxamine? Two approaches to overexpress VEGF in β-cell lines

Rapid and adequate revascularization of transplanted islets is important for their survival and function during transplantation. Vascular endothelial growth factor (VEGF) could play a critical role with respect to islet revascularization. The aim of this study was to compare two strategies that are used to overexpress VEGF in β-cells: (1) gene therapy through adenoviral infection and (2) a pharmacological approach using deferoxamine (DFO). β-Cell lines from rat insulinoma (RINm5F) were either infected using an adenovirus encoding the gene of human VEGF 165 or incubated with DFO. One day after treatment, the viability of RINm5F cells was preserved with 10 μmol/L of DFO (103.95 ± 5.66% toward control; n = 4). In addition, adenoviral infection maintained the viability of cells for all the concentrations used. In both treatments, overexpression of VEGF was in a comparable level. Finally, the ratio of Bax/Bcl-2 indicated that the apoptosis increased in infected β-cells whereas treatment with DFO seems to be antiapoptotic. Our results suggest that the use of DFO could be a realistic approach to improve the vascularization of islets during transplantation.