PURPOSE: Two major obstacles affect Pancreatic islet transplantation: the restricted availability of human donors and the need for life-long recipient’s general immunosuppression. These drawbacks could be solved by using neonatal porcine islets for human substitutes and by entrapping cells into multicompartmental alginate-poly L-ornithine- alginate (ALG-PLO-ALG) microcapsules (MC) supplied with systems enabling delivery of vitamins and enzymes, and growth factors. Sustained release of these actives could be used to treat post-transplant adverse events as well. Therefore, this work was aimed to determine the in vivo effect of SOD and KET controlled release from poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) microspheres (MS) on unexplained random in vivo host’s response to intraperitoneal grafting of ALG-PLO-ALG MC prepared with purified ALG. MATERIALS AND METHODS MS Preparation and characterization SOD loaded PLGA (Bidachem, Italy) MS and KET loaded PLA (Sigma, Milan, Italy) MS were prepared by a W/O/W double emulsion and an O/W emulsion solvent extraction/evaporation methods respectively. The protein content was determined by Micro-BCA protein assay (Sigma, Milan, Italy) by using a 8453 Agilent UV/VIS spectrophotometer (Agilent Technologies, Germany). KET content was determined by UV/VIS after dissolving the MS in methylene chloride. MS size distribution was determined by an Accusizer TM 770 Optical Particle Sizer (PSS Inc., Santa Barbara, CA, USA). Morphology was investigated by scanning electron microscopy (SEM) using a Philips XL30 microscope (Philips, the Netherlands). The entrapped SOD activity retention was evaluated in vitro according to the pyrogallol (Sigma, Milan, Italy) autoxidation inhibition assay. The in vitro SOD and KET release was determined in 0.1M pH 7.4 phosphate buffer, at 37°C. Samples were analyzed by Micro-BCA protein assay and UV. Alginate analysis and MC fabrication and characterization The purified ALG was assayed by the Bradford protein assay, the LAL (limulus amebocyte lysate) test at both the University of Perugia (Italy) and Living Cell Technologies laboratories (SOP Q209, Belgium) and for metals at Sereco Biotest s.p.a. (Perugia, Italy). MS inclusion into ALG/PLO/ALG MC was carried out according to a previously optimized method. SOD and KET release from ALG/PLO/ALG MC was performed in 0.1M pH 7.4 phosphate buffer at 37°C. PLO leakage from the MC was also investigated by Micro-BCA analysis. MC transplant and cell overgrowth analysis The experiment was performed according to a protocol previously approved by the Ethic Committee of the University of Perugia (p.n. 19382 PRE 805/2003). CD1 mice (average weight 25 g, Charles River Laboratories, Wilmington, MA), divided into (n=9) one control with conventional MC, one multicompartmental MC with SOD and one with KET, were terminated at day 10, 20, and 30. The recovered MC were examined by optical microscopy (Leica, Italy) for cell overgrowth and cells were detached from the MC and treated with rat anti-CD16/32 (2.4G2) and assayed by EPICS flow cytometer (Beckman Coulter). Rat anti-F4/80 and anti-CD8 or anti-CD11b, anti-CD25 and anti-CD69 were applied. RESULTS: Protein loading afforded 90-92% efficiency and 9% drug content. The mean volume diameter ranged between 13 and 18 µm. Span was 2.46. SOD activity within MS was successfully retained. KET loaded PLA MS showed the same characteristics previously recorded, with mean size around 5 µm and 99% of the population < 20 μm. Drug content and encapsulation efficiency were 17% (w/w) and 75%, respectively. SEM microphotographs highlighted a smooth surface and fairly regular shape for both MS formulations. Optical microscopy showed MC of rather regular and uniform shape and size. Both conventional and multicompartmental had the same features. All MC showed similar size around 450 µm. The MS distribution within the MC was homogeneous and no adhesion to the double coated ALG membrane was observed. SOD had a sustained release from PLGA MS with just 25% initial burst and the release reached 90% after 64 days in an almost linear manner (r2=0.993). SOD release from multicompartmental MC barely approached 40% at 30 days with a slow progression over time and stopped afterwards. KET release reached 90% after 7 days from PLA MS, while it dropped down to 70% from inside the MC. PLO was not released from the MC as no significant amounts (2-3% w/w) were found in the release medium up to 10-12 days. Although the purified ALG was considered biocompatible, the conventional MC collected at day 10, 20 and 30 were completely coated by cells. In turn, the MC containing KET became completely covered by cells only at day 20, while the MC with SOD did not show significant cell growth over the entire post-transplant follow-up. The profile of cell phenotypes recovered from the collected MC is summarized in Table 1. The control group showed, at day 10, 50% of F4/80+ peritoneal macrophages, which decreased to about 40% at day 30. 30% of the macrophages expressed a constant percentage of CD11b+ cells over time. The KET group presented 92% CD11b+ cells since day 10, which at day 30 decreased down to 51%, while the F4/80+ cells were 61%. The SOD group displayed, at day 20, 61% of F4/80+ cells, among which 48% expressed also for CD11b+, while, at day 30, a bare 8% of F4/80+ cells and no CD11b+ phenotype. T-cell expression at day 30 was about 19% CD8+ in the control, 6% in the KET and barely 4% in the SOD group. In the control group, 50% of CD8+ expressed an activated phenotype. CONCLUSIONS In spite of ALG of tested clinical-grade, host’s reactions to the material employed cannot be excluded. Therefore, the use of a combination of KET and SOD releasing MS is recommended to preserve the efficiency of grafts in the immediate and longer post-transplant follow-up and to warrant success of cell transplantation and survival.
EFFECT OF SOD AND KET RELEASE FROM BIODEGRADABLE MICROSPHERES ON THE IN VIVO RESPONSE TO IMPLANTED ALG-PLO-ALG MICROCAPSULES.
GIOVAGNOLI, Stefano;BLASI, PAOLO;LUCA, Giovanni;FALLARINO, Francesca;CALVITTI, Mario;MANCUSO, FRANCESCA;SCHOUBBEN, Aurelie Marie Madeleine;RICCI, Maurizio;BECCHETTI, Ennio;ROSSI, Carlo;CALAFIORE, Riccardo
2009
Abstract
PURPOSE: Two major obstacles affect Pancreatic islet transplantation: the restricted availability of human donors and the need for life-long recipient’s general immunosuppression. These drawbacks could be solved by using neonatal porcine islets for human substitutes and by entrapping cells into multicompartmental alginate-poly L-ornithine- alginate (ALG-PLO-ALG) microcapsules (MC) supplied with systems enabling delivery of vitamins and enzymes, and growth factors. Sustained release of these actives could be used to treat post-transplant adverse events as well. Therefore, this work was aimed to determine the in vivo effect of SOD and KET controlled release from poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) microspheres (MS) on unexplained random in vivo host’s response to intraperitoneal grafting of ALG-PLO-ALG MC prepared with purified ALG. MATERIALS AND METHODS MS Preparation and characterization SOD loaded PLGA (Bidachem, Italy) MS and KET loaded PLA (Sigma, Milan, Italy) MS were prepared by a W/O/W double emulsion and an O/W emulsion solvent extraction/evaporation methods respectively. The protein content was determined by Micro-BCA protein assay (Sigma, Milan, Italy) by using a 8453 Agilent UV/VIS spectrophotometer (Agilent Technologies, Germany). KET content was determined by UV/VIS after dissolving the MS in methylene chloride. MS size distribution was determined by an Accusizer TM 770 Optical Particle Sizer (PSS Inc., Santa Barbara, CA, USA). Morphology was investigated by scanning electron microscopy (SEM) using a Philips XL30 microscope (Philips, the Netherlands). The entrapped SOD activity retention was evaluated in vitro according to the pyrogallol (Sigma, Milan, Italy) autoxidation inhibition assay. The in vitro SOD and KET release was determined in 0.1M pH 7.4 phosphate buffer, at 37°C. Samples were analyzed by Micro-BCA protein assay and UV. Alginate analysis and MC fabrication and characterization The purified ALG was assayed by the Bradford protein assay, the LAL (limulus amebocyte lysate) test at both the University of Perugia (Italy) and Living Cell Technologies laboratories (SOP Q209, Belgium) and for metals at Sereco Biotest s.p.a. (Perugia, Italy). MS inclusion into ALG/PLO/ALG MC was carried out according to a previously optimized method. SOD and KET release from ALG/PLO/ALG MC was performed in 0.1M pH 7.4 phosphate buffer at 37°C. PLO leakage from the MC was also investigated by Micro-BCA analysis. MC transplant and cell overgrowth analysis The experiment was performed according to a protocol previously approved by the Ethic Committee of the University of Perugia (p.n. 19382 PRE 805/2003). CD1 mice (average weight 25 g, Charles River Laboratories, Wilmington, MA), divided into (n=9) one control with conventional MC, one multicompartmental MC with SOD and one with KET, were terminated at day 10, 20, and 30. The recovered MC were examined by optical microscopy (Leica, Italy) for cell overgrowth and cells were detached from the MC and treated with rat anti-CD16/32 (2.4G2) and assayed by EPICS flow cytometer (Beckman Coulter). Rat anti-F4/80 and anti-CD8 or anti-CD11b, anti-CD25 and anti-CD69 were applied. RESULTS: Protein loading afforded 90-92% efficiency and 9% drug content. The mean volume diameter ranged between 13 and 18 µm. Span was 2.46. SOD activity within MS was successfully retained. KET loaded PLA MS showed the same characteristics previously recorded, with mean size around 5 µm and 99% of the population < 20 μm. Drug content and encapsulation efficiency were 17% (w/w) and 75%, respectively. SEM microphotographs highlighted a smooth surface and fairly regular shape for both MS formulations. Optical microscopy showed MC of rather regular and uniform shape and size. Both conventional and multicompartmental had the same features. All MC showed similar size around 450 µm. The MS distribution within the MC was homogeneous and no adhesion to the double coated ALG membrane was observed. SOD had a sustained release from PLGA MS with just 25% initial burst and the release reached 90% after 64 days in an almost linear manner (r2=0.993). SOD release from multicompartmental MC barely approached 40% at 30 days with a slow progression over time and stopped afterwards. KET release reached 90% after 7 days from PLA MS, while it dropped down to 70% from inside the MC. PLO was not released from the MC as no significant amounts (2-3% w/w) were found in the release medium up to 10-12 days. Although the purified ALG was considered biocompatible, the conventional MC collected at day 10, 20 and 30 were completely coated by cells. In turn, the MC containing KET became completely covered by cells only at day 20, while the MC with SOD did not show significant cell growth over the entire post-transplant follow-up. The profile of cell phenotypes recovered from the collected MC is summarized in Table 1. The control group showed, at day 10, 50% of F4/80+ peritoneal macrophages, which decreased to about 40% at day 30. 30% of the macrophages expressed a constant percentage of CD11b+ cells over time. The KET group presented 92% CD11b+ cells since day 10, which at day 30 decreased down to 51%, while the F4/80+ cells were 61%. The SOD group displayed, at day 20, 61% of F4/80+ cells, among which 48% expressed also for CD11b+, while, at day 30, a bare 8% of F4/80+ cells and no CD11b+ phenotype. T-cell expression at day 30 was about 19% CD8+ in the control, 6% in the KET and barely 4% in the SOD group. In the control group, 50% of CD8+ expressed an activated phenotype. CONCLUSIONS In spite of ALG of tested clinical-grade, host’s reactions to the material employed cannot be excluded. Therefore, the use of a combination of KET and SOD releasing MS is recommended to preserve the efficiency of grafts in the immediate and longer post-transplant follow-up and to warrant success of cell transplantation and survival.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.