Immune-Protective Formulations and Process Strategies for Improved Survival and Function of Transplanted Islets.

Type 1 diabetes (T1D) is an autoimmune disease caused by the immune system attacking and destroying insulin-producing ß cells in the pancreas. Islet transplantation is becoming one of the most promising therapies for T1D patients. However, its clinical use is limited by substantial cell loss after islet infusion, closely related to immune reactions, including instant blood-mediated inflammatory responses, oxidative stress, and direct autoimmune attack. Especially the grafted islets are not only exposed to allogeneic immune rejection after transplantation but are also subjected to an autoimmune process that caused the original disease. Due to the development and convergence of expertise in biomaterials, nanotechnology, and immunology, protective strategies are being investigated to address this issue, including exploring novel immune protective agents, encapsulating islets with biomaterials, and searching for alternative implantation sites, or co-transplantation with functional cells. These methods have significantly increased the survival rate and function of the transplanted islets. However, most studies are still limited to animal experiments and need further studies. In this review, we introduced the immunological challenges for islet graft and summarized the recent developments in immune-protective strategies to improve the outcomes of islet transplantation.

Engineering ß Cell Replacement Therapies for Type 1 Diabetes: Biomaterial Advances and Considerations for Macroscale Constructs.

While significant progress has been made in treatments for type 1 diabetes (T1D) based on exogenous insulin, transplantation of insulin-producing cells (islets or stem cell-derived ß cells) remains a promising curative strategy. The current paradigm for T1D cell therapy is clinical islet transplantation (CIT)-the infusion of islets into the liver-although this therapeutic modality comes with its own limitations that deteriorate islet health. Biomaterials can be leveraged to actively address the limitations of CIT, including undesired host inflammatory and immune responses, lack of vascularization, hypoxia, and the absence of native islet extracellular matrix cues. Moreover, in efforts toward a clinically translatable T1D cell therapy, much research now focuses on developing biomaterial platforms at the macroscale, at which implanted platforms can be easily retrieved and monitored. In this review, we discuss how biomaterials have recently been harnessed for macroscale T1D ß cell replacement therapies.

A Zwitterionic Polyurethane Nanoporous Device with Low Foreign-Body Response for Islet Encapsulation.

Encapsulation of insulin-producing cells is a promising strategy for treatment of type 1 diabetes. However, engineering an encapsulation device that is both safe (i.e., no cell escape and no breakage) and functional (i.e., low foreign-body response (FBR) and high mass transfer) remains a challenge. Here, a family of zwitterionic polyurethanes (ZPU) with sulfobetaine groups in the polymer backbone is developed, which are fabricated into encapsulation devices with tunable nanoporous structures via electrospinning. The ZPU encapsulation device is hydrophilic and fouling-resistant, exhibits robust mechanical properties, and prevents cell escape while still allowing efficient mass transfer. The ZPU device also induces a much lower FBR or cellular overgrowth upon intraperitoneal implantation in C57BL/6 mice for up to 6 months compared to devices made of similar polyurethane without the zwitterionic modification. The therapeutic potential of the ZPU device is shown for islet encapsulation and diabetes correction in mice for ≈3 months is demonstrated. As a proof of concept, the scalability and retrievability of the ZPU device in pigs and dogs are further demonstrated. Collectively, these attributes make ZPU devices attractive candidates for cell encapsulation therapies.

Immunosuppressive PLGA TGF-ß1 Microparticles Induce Polyclonal and Antigen-Specific Regulatory T Cells for Local Immunomodulation of Allogeneic Islet Transplants.

Allogeneic islet transplantation is a promising cell-based therapy for Type 1 Diabetes (T1D). The long-term efficacy of this approach, however, is impaired by allorejection. Current clinical practice relies on long-term systemic immunosuppression, leading to severe adverse events. To avoid these detrimental effects, poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were engineered for the localized and controlled release of immunomodulatory TGF-ß1. The in vitro co-incubation of TGF-ß1 releasing PLGA MPs with naïve CD4+ T cells resulted in the efficient generation of both polyclonal and antigen-specific induced regulatory T cells (iTregs) with robust immunosuppressive function. The co-transplantation of TGF-ß1 releasing PLGA MPs and Balb/c mouse islets within the extrahepatic epididymal fat pad (EFP) of diabetic C57BL/6J mice resulted in the prompt engraftment of the allogenic implants, supporting the compatibility of PLGA MPs and local TGF-ß1 release. The presence of the TGF-ß1-PLGA MPs, however, did not confer significant graft protection when compared to untreated controls, despite measurement of preserved insulin expression, reduced intra-islet CD3+ cells invasion, and elevated CD3+Foxp3+ T cells at the peri-transplantation site in long-term functioning grafts. Examination of the broader impacts of TGF-ß1/PLGA MPs on the host immune system implicated a localized nature of the immunomodulation with no observed systemic impacts. In summary, this approach establishes the feasibility of a local and modular microparticle delivery system for the immunomodulation of an extrahepatic implant site. This approach can be easily adapted to deliver larger doses or other agents, as well as multi-drug approaches, within the local graft microenvironment to prevent transplant rejection.

Experience with a novel efalizumab-based immunosuppressive regimen to facilitate single donor islet cell transplantation.

Islet transplantation is an experimental therapy for selected patients with type 1 diabetes (T1DM). It remains limited by immunosuppressive drug toxicity, progressive loss of insulin independence, allosensitization and the need for multiple islet donors. We describe our experience with an efalizumab-based immunosuppressive regimen as compared to the prevailing standard regimen, the Edmonton protocol. Twelve patients with T1DM received islet transplants: eight were treated with the Edmonton protocol; four were treated with daclizumab induction, a 6-month course of tacrolimus, and maintenance with efalizumab and mycophenolate mofetil. The primary endpoint was insulin independence after one islet infusion. Only two Edmonton protocol treated patients achieved the primary endpoint; six required islets from multiple donors, and all experienced leukopenia, mouth ulcers, anemia, diarrhea and hypertransaminasemia. Four became allosensitized. All patients treated with the efalizumab-based regimen achieved insulin independence with normal hemoglobin A1c after a single islet cell infusion and remained insulin independent while on efalizumab. These patients experienced significantly fewer side effects and none became allosensitized. Trial continuation was terminated by withdrawal of efalizumab from the market. These data suggest that this efalizumab-based regimen prevents islet rejection, is well tolerated, and allows for single donor islet transplantation.

Hepatic artery vs. portal vein infusion of microbeads: a large animal pre-clinical model evaluating the intrahepatic capacity for cell infusion and imaging.

BACKGROUND: Islet xeno-transplantation via the portal vein has been proposed as an alternative of islet allo-transplantation for treatment of type 1 diabetes. However, the precise hepatic capacity has not been addressed. METHODS: We mimicked cell transplantation by infusion of dogs with alginate/poly-1-lysine/alginate (APA) microbeads via either the portal vein (PV) or hepatic artery (HA). The maximal adaptable capacity for infused microbeads was evaluated by examination of vasculature, microvasculature, hepatic hemodynamics, portal vein, and hepatic artery pressures and liver function in the microbead recipient dogs. RESULTS: PV but not HA dogs demonstrated elevated portal pressure during the infusion procedure in a dose-dependent manner. Four out of twelve PV dogs infused with 32,000 microbeads/kg developed acute liver infarction within 24 h after infusion with four of the remaining eight animals developing portal venous thrombosis within 24 h following infusion. All PV animals demonstrated abnormal alanine aminotransferase (ALT) values, and the extent and duration of increased ALT levels correlated with the increase in the number of microbeads infused. In contrast, HA animals infused with as many as 32,000 microbeads/kg had neither portal thrombosis nor abnormal liver function. CONCLUSIONS: The capacity for intrahepatic cell infusion is finite and the intrahepatic artery may have a less hemodynamic interference impact on transplantation of cells into the liver when a larger volume of cells is required to achieve curable outcomes in both allo- and xeno-transplantation.

VEGF-Modified PVA/Silicone Nanofibers Enhance Islet Function Transplanted in Subcutaneous Site Followed by Device-Less Procedure.

INTRODUCTION: As heterologous islets or islet-like stem cells become optional sources for islet transplantation, the subcutaneous site appears to be an acceptable replacement of the intrahepatic site due to its graft retrievability. The device-less (DL) procedure improves the feasibility; however, some limitations such as fibrotic overgrowth or immunodeficiency still exist. Nanofibers could mimic the extracellular matrix to improve the vitality of transplanted islets. Therefore, we designed a vascular endothelial growth factor (VEGF)-modified polyvinyl alcohol (PVA)/silicone nanofiber (SiO2-VEGF) to optimize the DL procedure. METHODS: SiO2-VEGF nanofibers were designed by nano-spinning and characterized the physical-chemical properties before subcutaneous islet transplantation. Cell viability, vessel formation, and glucose-stimulated insulin secretion were tested in vitro to ensure biocompatibility; and blood glucose level (BGL), transplanted islet function, and epithelial-mesenchymal transition (EMT)-related biomarker expression were analyzed in vivo. RESULTS: The intensity of inflammatory reaction induced by SiO2 nanofibers was between nylon and silicone, which did not bring out excessive fibrosis. The vascularization could be enhanced by VEGF functionalization both in vitro and in vivo. The BGL control was better in the DL combined with SiO2-VEGF group. The percentage of recipients that achieved normoglycemia was higher and earlier (71% at day 57), and the intraperitoneal glucose tolerance test (IPGTT) also confirmed better islet function. The expressions of vimentin, α-SMA, and twist-1 were upregulated, which indicated that SiO2-VEGF nanofibers might promote islet function by regulating the EMT pathway. DISCUSSION: In summary, our new SiO2-VEGF combined with DL procedure might improve the feasibility of subcutaneous islet transplantation for clinical application.

Overcoming foreign-body reaction through nanotopography: Biocompatibility and immunoisolation properties of a nanofibrous membrane.

Implantable immunoisolation membranes need to possess superior biocompatibility to prohibit the fibrotic deposition that would reduce the nutrient supply and impair the viability/function of the encapsulated cells. Here, electrospun membranes based on thermoplastic polyurethane (TPU) were fabricated to contain microfibers (PU-micro) or nanofibers (PU-nano). The two types of membranes were compared in terms of their interaction with macrophage cells and the host tissues. It was found that the fibrous membranes of different topographies possess distinct material properties: PU-nano caused minimal macrophage responses in vitro and in vivo and induced only mild foreign body reactions compared to PU-micro membranes. A flat macroencapsulation device was fabricated using PU-nano membranes and its immunoisolation function investigated in subcutaneous transplantation models. The nanofibrous device demonstrated the capability to effectively shield the allografts from the immune attack of the host. Nanotopography may confer biocompatibility to materials and nanofibrous materials warrant further study for development of "invisible" immunoisolation devices for cell transplantation.

Long-term Efficacy and Biocompatibility of Encapsulated Islet Transplantation With Chitosan-Coated Alginate Capsules in Mice and Canine Models of Diabetes.

BACKGROUND: Clinical application of encapsulated islet transplantation is hindered by low biocompatibility of capsules leading to pericapsular fibrosis and decreased islet viability. To improve biocompatibility, we designed a novel chitosan-coated alginate capsules and compared them to uncoated alginate capsules. METHODS: Alginate capsules were formed by crosslinking with BaCl2, then they were suspended in chitosan solution for 10 minutes at pH 4.5. Xenogeneic islet transplantation, using encapsulated porcine islets in 1,3-galactosyltransferase knockout mice, and allogeneic islet transplantation, using encapsulated canine islets in beagles, were performed without immunosuppressants. RESULTS: The chitosan-alginate capsules showed similar pore size, islet viability, and insulin secretory function compared to alginate capsules, in vitro. Xenogeneic transplantation of chitosan-alginate capsules demonstrated a trend toward superior graft survival (P = 0.07) with significantly less pericapsular fibrosis (cell adhesion score: 3.77 ± 0.41 vs 8.08 ± 0.05; P < 0.001) compared to that of alginate capsules up to 1 year after transplantation. Allogeneic transplantation of chitosan-alginate capsules normalized the blood glucose level up to 1 year with little evidence of pericapsular fibrotic overgrowth on graft explantation. CONCLUSIONS: The efficacy and biocompatibility of chitosan-alginate capsules were demonstrated in xenogeneic and allogeneic islet transplantations using small and large animal models of diabetes. This capsule might be a potential candidate applicable in the treatment of type 1 diabetes mellitus patients, and further studies in nonhuman primates are required.

Bioengineering the Endocrine Pancreas: Intraomental Islet Transplantation Within a Biologic Resorbable Scaffold.

Transplantation of pancreatic islets is a therapeutic option to preserve or restore ß-cell function. Our study was aimed at developing a clinically applicable protocol for extrahepatic transplantation of pancreatic islets. The potency of islets implanted onto the omentum, using an in situ-generated adherent, resorbable plasma-thrombin biologic scaffold, was evaluated in diabetic rat and nonhuman primate (NHP) models. Intraomental islet engraftment in the biologic scaffold was confirmed by achievement of improved metabolic function and preservation of islet cytoarchitecture, with reconstitution of rich intrainsular vascular networks in both species. Long-term nonfasting normoglycemia and adequate glucose clearance (tolerance tests) were achieved in both intrahepatic and intraomental sites in rats. Intraomental graft recipients displayed lower levels of serum biomarkers of islet distress (e.g., acute serum insulin) and inflammation (e.g., leptin and α2-macroglobulin). Importantly, low-purity (30:70% endocrine:exocrine) syngeneic rat islet preparations displayed function equivalent to that of pure (>95% endocrine) preparations after intraomental biologic scaffold implantation. Moreover, the biologic scaffold sustained allogeneic islet engraftment in immunosuppressed recipients. Collectively, our feasibility/efficacy data, along with the simplicity of the procedure and the safety of the biologic scaffold components, represented sufficient preclinical testing to proceed to a pilot phase I/II clinical trial.

Ketoprofen controlled release from composite microcapsules for cell encapsulation: effect on post-transplant acute inflammation.

Cell encapsulation technology raises hopes in medicine and biotechnology. Encapsulated pancreatic islets is a promising approach for the final solution of Type 1 diabetes. Unfortunately, evidence of long-term encapsulated islet graft survival and functional competence lies behind expectancy. Failure was often ascribed to the lack of biocompatibility generating inflammatory response, or limited immunobarrier competence or hypoxia or finally, low beta-cell replication. In order to prevent severe inflammation at early stages after implantation, composite microcapsules were designed. Biodegradable microspheres containing ketoprofen were enveloped into the well established alginate/poly-L-ornithine/alginate capsules. Polyester microspheres were prepared, by solvent evaporation, and characterized for encapsulation efficiency, particle size and in vitro release. Biocompatibility and efficacy to prevent the inflammatory response were studied in vivo. Good encapsulation efficiency and the desired particle size were achieved. In vitro release studies evidenced a high burst effect probably due to a plasticizing effect of both water and ketoprofen. The composite systems showed good biocompatibility and capacity to completely avoid the inflammatory response and the pericapsular cell overgrowth. In conclusion, the inflammatory response in the immediate post-transplant period can be circumvented using multicompartment microcapsules releasing non-steroidal anti inflammatory drugs.

Benefits and risks of solitary islet transplantation for type 1 diabetes using steroid-sparing immunosuppression: the National Institutes of Health experience.

OBJECTIVE: The aim of this study was to describe the National Institutes of Health's experience initiating an islet isolation and transplantation center, including descriptions of our first six recipients, and lessons learned. RESEARCH DESIGN AND METHODS: Six females with chronic type 1 diabetes, hypoglycemia unawareness, and no endogenous insulin secretion (undetectable serum C-peptide) were transplanted with allogenic islets procured from brain dead donors. To prevent islet rejection, patients received daclizumab, sirolimus, and tacrolimus. RESULTS: All patients noted less frequent and less severe hypoglycemia, and one-half were insulin independent at 1 year. Serum C-peptide persists in all but one patient (follow-up 17-22 months), indicating continued islet function. Two major procedure-related complications occurred: partial portal vein thrombosis and intra-abdominal hemorrhage. While we observed no cytomegalovirus infection or malignancy, recipients frequently developed transient mouth ulcers, diarrhea, edema, hypercholesterolemia, weight loss, myelosuppression, and other symptoms. Three patients discontinued immunosuppressive therapy: two because of intolerable toxicity (deteriorating kidney function and sirolimus-induced pneumonitis) while having evidence for continued islet function (one was insulin independent) and one because of gradually disappearing islet function. CONCLUSIONS: We established an islet isolation and transplantation program and achieved a 50% insulin-independence rate after at most two islet infusions. Our experience demonstrates that centers not previously engaged in islet transplantation can initiate a program, and our data and literature analysis support not only the promise of islet transplantation but also its remaining hurdles, which include the limited islet supply, procedure-associated complications, imperfect immunosuppressive regimens, suboptimal glycemia control, and loss of function over time.

Molecular imaging monitoring of poly(ethylene glycol) conjugated islets for evaluation of islet graft rejection.

It is important to chase the function of islet after transplantation. Thus, we examined the correlation between grafted islets function and molecular imaging intensity using Cy5.5 labeled islet. The ability of Cy5.5-PEG-NHS to chemically bind on the surface of islets was determined by confocal laser scanning microscope. Then, fluorescence intensity of different number of Cy5.5 labeled islets was determined using optical imaging system. We have found out the intensity of fluorescence increased with increasing the total number of islet in each well. In addition, different number of Cy5.5-labeled islet has been transplanted into the athymic mice for in vivo imaging. The intensity emitted from Cy5.5-labeled islets augmented proportionally with increased number of transplanted islets. To understand the correlation between the function of grafted islet and the fluorescence intensity emitted optical imaging system, Cy5.5-labeled islets have been transplanted into F344 rats. The results revealed that there was a correlation between the fluorescence intensity and the non-fasting blood glucose (NBG) levels of islets received rats. Strong fluorescence intensity corresponded to low NBG whereas low signal was associated to high NBG. In conclusion, the fluorescence intensity emitted from Cy5.5-labeled islets can be used as a marker of cells viability and functionality after transplantation.

Immune reactions of lymphocytes and macrophages against PEG-grafted pancreatic islets.

Graft rejection is the major limiting factor in islet transplantation and is closely related with the recruitment and activation of T cells and macrophages against the graft. To reduce the immunogenicity of islets, we have grafted biocompatible polyethylene glycol (PEG) onto the collagen capsule of islets without changing the morphology and function of islets. In this study, we evaluated whether the grafted PEG molecules on the collagen capsule of islet could prevent the activation of immune cells, and investigated factors that are mainly related to the immune reaction in vitro. During the co-culture with lymphocytes, the morphology and viability of PEG-grafted islets were not damaged, and the amounts of IL-2 and TNF-alpha secreted from lymphocytes co-cultured with PEG-grafted islets were significantly lower than that of free islets. However, when both kinds of islets were cultured with macrophages, there were no significant differences in morphology, viability and the secreted amounts of cytokines and nitric oxide. In conclusion, the grafted PEG could inhibit activation of lymphocytes, which are essential in initiating the graft rejection process. However, the grafted PEG molecules could not completely prevent the infiltration of cytotoxic molecules into the islets.

Islet and stem cell encapsulation for clinical transplantation.

Over the last decade, improvements in islet isolation techniques have made islet transplantation an option for a certain subset of patients with long-standing diabetes. Although islet transplants have shown improved graft function, adequate function beyond the second year has not yet been demonstrated, and patients still require immunosuppression to prevent rejection. Since allogeneic islet transplants have experienced some success, the next step is to improve graft function while eliminating the need for systemic immunosuppressive therapy. Biomaterial encapsulation offers a strategy to avoid the need for toxic immunosuppression while increasing the chances of graft function and survival. Encapsulation entails coating cells or tissue in a semipermeable biocompatible material that allows for the passage of nutrients, oxygen, and hormones while blocking immune cells and regulatory substances from recognizing and destroying the cell, thus avoiding the need for systemic immunosuppressive therapy. Despite advances in encapsulation technology, these developments have not yet been meaningfully translated into clinical islet transplantation, for which several factors are to blame, including graft hypoxia, host inflammatory response, fibrosis, improper choice of biomaterial type, lack of standard guidelines, and post-transplantation device failure. Several new approaches, such as the use of porcine islets, stem cells, development of prevascularized implants, islet nanocoating, and multilayer encapsulation, continue to generate intense scientific interest in this rapidly expanding field. This review provides a comprehensive update on islet and stem cell encapsulation as a treatment modality in type 1 diabetes, including a historical outlook as well as current and future research avenues.

Efficacy of DHMEQ, a NF-κB inhibitor, in islet transplantation: I. HMGB1 suppression by DHMEQ prevents early islet graft damage.

BACKGROUND: Pancreatic islet transplantation (PITx) is an attractive treatment option for restoring appropriate glucose homeostasis in type 1 diabetes patients. Although islet grafts can successfully engraft after PITx, large numbers of islet grafts are required mainly because immune reactions, including inflammation, destroy islet grafts. In these processes, nuclear factor (NF)-κB plays a central role. We hypothesized that the inhibition of NF-κB activation would ameliorate inflammatory responses after PITx and aid successful engraftment. METHODS: To test this hypothesis, a newly developed NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), was used on a syngeneic mouse PITx model. One hundred seventy-five islets from C57BL/6 (B6) mice were transplanted into streptozotocin-induced diabetic B6 mice. The recipient mice were administered DHMEQ for 1, 2, or 3 days after PITx. The underlying mechanisms of DHMEQ on islet graft protection were investigated in an in vitro coculture model of pancreatic islets and macrophages. RESULTS: With a vehicle treatment, only 11.1% of the islet-recipients achieved normoglycemia after PITx. In sharp contrast, DHMEQ treatment markedly improved the normoglycemic rate, which was associated with the suppression of serum high mobility group complex-1 (HMGB1) and proinflammatory cytokines, including tumor necrosis factor-α, monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, interleukin-1ß, and interleukin-6, after PITx. In a murine macrophage-like cell line, DHMEQ inhibited HMGB1-driven activation and proinflammatory cytokine secretion and further prevented death isolated islets after coculture with these activated macrophages. CONCLUSIONS: Inhibition of NF-κB activation by DHMEQ after PITx reduces the HMGB1-triggered proinflammatory responses and results in engraftment of transplanted islets even with fewer islet grafts.

Risks and side effects of islet transplantation.

Islet transplantation can deliver stable glycemic control, relief from recurrent severe hypoglycemia, and insulin independence. Accessing the portal vein via the percutaneous hepatic approach carries the risk of bleeding, and the infusion of islets a risk of portal vein thrombosis. In the long term, common minor problems with immunosuppression are mouth ulcers, diarrhea, and acne. Longer-term risks include malignancy and serious infection, both rare to date in clinical islet transplantation. Sensitization to donor antigens may also occur. The long-term diabetes complications may stabilize, but of this aspect little is known to date. In the short term, there may be some elevation of serum cholesterol and blood pressure, in some patients there has been a decline in renal function, and in a few, acute retinal bleeds. For most, improvement in glucose control with resolution of glycemic lability and hypoglycemia has been a net benefit.