In vitro evaluation of a hybrid drug-delivery nanosystem for fibrosis prevention in cell therapy for Type 1 diabetes.

Background: Implantation of insulin-secreting cells has been trialed as a treatment for Type 1 diabetes mellitus; however, the host immunogenic response limits their effectiveness. Methodology: The authors developed a core-shell nanostructure of upconversion nanoparticle-mesoporous silica for controlled local delivery of an immunomodulatory agent, MCC950, using near-infrared light and validated it in in vitro models of fibrosis. Results: The individual components of the nanosystem did not affect the proliferation of insulin-secreting cells, unlike fibroblast proliferation (p < 0.01). The nanosystem is effective at releasing MCC950 and preventing fibroblast differentiation (p < 0.01), inflammation (IL-6 expression; p < 0.05) and monocyte adhesion (p < 0.01). Conclusion: This MCC950-loaded nanomedicine system could be used in the future together with insulin-secreting cell implants to increase their longevity as a curative treatment for Type 1 diabetes mellitus.

This work describes a new drug-delivery system that can release an immunomodulatory drug in a controlled manner and prevent fibrosis, which is part of the immune response when a foreign body is implanted. This system can be particularly useful for insulin-secreting cell implants, used to replace multiple daily injections of insulin and improve the quality of life of people with Type 1 diabetes mellitus. By preventing the immune response that leads to fibrosis, the longevity of these cellular implants can be extended without the need for frequent replacement procedures. This innovative nanosystem can release the required amount of immunomodulatory drug, which could be stimulated with the use of special light, hence showing the ability for local and extended delivery. This type of system has the potential to reduce the side effects associated with oral daily administration of immunomodulatory agents in people with Type 1 diabetes mellitus.

Challenges with Cell-based Therapies for Type 1 Diabetes Mellitus.

Type 1 diabetes (T1D) is a chronic, lifelong metabolic disease. It is characterised by the autoimmune-mediated loss of insulin-producing pancreatic ß cells in the islets of Langerhans (ß-islets), resulting in disrupted glucose homeostasis. Administration of exogenous insulin is the most common management method for T1D, but this requires lifelong reliance on insulin injections and invasive blood glucose monitoring. Replacement therapies with beta cells are being developed as an advanced curative treatment for T1D. Unfortunately, this approach is limited by the lack of donated pancreatic tissue, the difficulties in beta cell isolation and viability maintenance, the longevity of the transplanted cells in vivo, and consequently high costs. Emerging approaches to address these limitations are under intensive investigations, including the production of insulin-producing beta cells from various stem cells, and the development of bioengineered devices including nanotechnologies for improving islet transplantation efficacy without the need for recipients taking toxic anti-rejection drugs. These emerging approaches present promising prospects, while the challenges with the new techniques need to be tackled for ultimately clinical treatment of T1D. This review discussed the benefits and limitations of the cell-based therapies for beta cell replacement as potential curative treatment for T1D, and the applications of bioengineered devices including nanotechnology to overcome the challenges associated with beta cell transplantation.

Cell delivery systems: Toward the next generation of cell therapies for type 1 diabetes.

Immunoprotection and oxygen supply are vital in implementing a cell therapy for type 1 diabetes (T1D). Without these features, the transplanted islet cell clusters will be rejected by the host immune system, and necrosis will occur due to hypoxia. The use of anti-rejection drugs can help protect the transplanted cells from the immune system; yet, they also may have severe side effects. Cell delivery systems (CDS) have been developed for islet transplantation to avoid using immunosuppressants. CDS provide physical barriers to reduce the immune response and chemical coatings to reduce host fibrotic reaction. In some CDS, there is architecture to support vascularization, which enhances oxygen exchange. In this review, we discuss the current clinical and preclinical studies using CDS without immunosuppression as a cell therapy for T1D. We find that though CDS have been demonstrated for their ability to support immunoisolation of the grafted cells, their functionality has not been fully optimized. Current advanced methods in clinical trials demonstrate the systems are partly functional, physically complicated to implement or inefficient. However, modifications are being made to overcome these issues.

Prevascularized Retrievable Hybrid Implant to Enhance Function of Subcutaneous Encapsulated Islets.

Replacement of pancreatic ß-cells is one of the most promising treatment options for treatment of type 1 diabetes (T1D), even though, toxic immunosuppressive drugs are required. In this study, we aim to deliver allogeneic ß-cell therapies without antirejection drugs using a bioengineered hybrid device that contains microencapsulated ß-cells inside 3D polycaprolactone (PCL) scaffolds printed using melt electrospin writing (MEW). Mouse ß-cell (MIN6) pseudoislets and QS mouse islets are encapsulated in alginate microcapsules, without affecting viability and insulin secretion. Microencapsulated MIN6 cells are then seeded within 3D MEW scaffolds, and these hybrid devices implanted subcutaneously in streptozotocin-treated diabetic NOD/SCID and BALB/c mice. Similar to NOD/SCID mice, blood glucose levels (BGL) are lowered from 30.1 to 4.8 mM in 25-41 days in BALB/c. In contrast, microencapsulated islets placed in prevascularized MEW scaffold 3 weeks after implantation in BALB/c mice normalize BGL (<12 mM) more rapidly, lasting for 60-105 days. The lowering of glucose levels is confirmed by an intraperitoneal glucose tolerance test. Vascularity within the implanted grafts is demonstrated and quantified by 3D-doppler ultrasound, with a linear increase over 4 weeks (r = 0.65). Examination of the device at 5 weeks shows inflammatory infiltrates of neutrophils, macrophages, and B-lymphocytes on the MEW scaffolds, but not on microcapsules, which have infrequent profibrotic walling. In conclusion, we demonstrate the fabrication of an implantable and retrievable hybrid device for vascularization and enhancing the survival of encapsulated islets implanted subcutaneously in an allotransplantation setting without immunosuppression. This study provides proof-of-concept for the application of such devices for human use, but, will require modifications to allow translation to people with T1D. Impact statement The retrievable 3D printed PCL scaffold we have produced promotes vascularization when implanted subcutaneously and allows seeded microencapsulated insulin-producing cells to normalize blood glucose of diabetic mice for at least 2 months, without the need for antirejection drugs to be administered. The scaffold is scalable for possible human use, but will require modification to ensure that normalization of blood glucose levels can be maintained long term.

Smart Drug-Delivery System of Upconversion Nanoparticles Coated with Mesoporous Silica for Controlled Release.

Drug-delivery vehicles have garnered immense interest in recent years due to unparalleled progress made in material science and nanomedicine. However, the development of stimuli-responsive devices with controllable drug-release systems (DRSs) is still in its nascent stage. In this paper, we designed a two-way controlled drug-release system that can be promoted and prolonged, using the external stimulation of near-infrared light (NIR) and protein coating. A hierarchical nanostructure was fabricated using upconversion nanoparticles (UCNPs)-mesoporous silica as the core-shell structure with protein lysozyme coating. The mesoporous silica shell provides abundant pores for the loading of drug molecules and a specific type of photosensitive molecules. The morphology and the physical properties of the nanostructures were thoroughly characterized. The results exhibited the uniform core-shell nanostructures of ~four UCNPs encapsulated in one mesoporous silica nanoparticle. The core-shell nanoparticles were in the spherical shape with an average size of 200 nm, average surface area of 446.54 m2/g, and pore size of 4.6 nm. Using doxorubicin (DOX), a chemotherapy agent as the drug model, we demonstrated that a novel DRS with capacity of smart modulation to promote or inhibit the drug release under NIR light and protein coating, respectively. Further, we demonstrated the therapeutic effect of the designed DRSs using breast cancer cells. The reported novel controlled DRS with dual functionality could have a promising potential for chemotherapy treatment of solid cancers.

Encapsulation and Transplantation of Pancreatic Progenitor Cells.

Type 1 diabetes, characterized by autoimmune destruction of pancreatic beta cells, affects 41 million people worldwide. Beta cell replacement therapies have immense potential as a treatment option because pancreatic progenitors derived from human pluripotent stem cells can provide a near limitless supply of transplantable tissue. The key limitation of this approach is the need for lifelong use of immunosuppressive drugs that have undesirable side effects. Microencapsulation is an option for providing protection for transplanted cells from mechanical stress and immune attack. Traditionally, pluripotent cells are differentiated on a 2D matrix before being transferred into an immunoisolation device. Here, we describe a method of differentiating pluripotent stem cells into pancreatic progenitors while the cells are encapsulated in alginate microspheres. This method provides several advantages including the need for fewer steps compared to the traditional approach, protection against mechanical/physical damage during differentiation in bioreactors, and immune-protection of cells once transplanted into the host.

Australian Public Attitudes on Gene Editing of the Human Embryo.

Technology is now available which facilitates gene editing and has recently been applied internationally to embryos in the laboratory. A 2002 law in Australia prohibits making heritable changes in embryos, regardless of whether the treated embryo is discarded thereafter. We sought to begin to understand public opinion in Australia about this matter, using a questionnaire given to the audience attending a Q and A panel of experts. We found majority support for allowing heritable changes for health purposes. If this is confirmed in a larger survey of the population, we suggest the existing law should be reviewed.

Co-encapsulation and co-transplantation of mesenchymal stem cells reduces pericapsular fibrosis and improves encapsulated islet survival and function when allografted.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated islets. A strategy to combat PFO is the use of mesenchymal stem cells (MSC). MSC have anti-inflammatory properties and their potential can be enhanced by stimulation with proinflammatory cytokines. This study investigated whether co-encapsulation or co-transplantation of MSC with encapsulated islets would reduce PFO and improve graft survival. Stimulating MSC with a cytokine cocktail of IFN-γ and TNF-α enhanced their immunosuppressive potential by increasing nitric oxide production and secreting higher levels of immunomodulatory cytokines. In vitro, co-encapsulation with MSC did not affect islet viability but significantly enhanced glucose-induced insulin secretion. In vivo, normoglycemia was achieved in 100% mice receiving islets co-encapsulated with stimulated MSC as opposed to 71.4% receiving unstimulated MSC and only 9.1% receiving encapsulated islets alone. Microcapsules retrieved from both unstimulated and stimulated MSC groups had significantly less PFO with improved islet viability and function compared to encapsulated islets alone. Levels of peritoneal immunomodulatory cytokines IL-4, IL-6, IL-10 and G-CSF were significantly higher in MSC co-encapsulated groups. Similar results were obtained when encapsulated islets and MSC were co-transplanted. In summary, co-encapsulation or co-transplantation of MSC with encapsulated islets reduced PFO and improved the functional outcome of allotransplants.

Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

Encapsulated Islet Transplantation: Where Do We Stand?

Transplantation of pancreatic islets encapsulated within immuno-protective microcapsules is a strategy that has the potential to overcome graft rejection without the need for toxic immunosuppressive medication. However, despite promising preclinical studies, clinical trials using encapsulated islets have lacked long-term efficacy, and although generally considered clinically safe, have not been encouraging overall. One of the major factors limiting the long-term function of encapsulated islets is the host's immunological reaction to the transplanted graft which is often manifested as pericapsular fibrotic overgrowth (PFO). PFO forms a barrier on the capsule surface that prevents the ingress of oxygen and nutrients leading to islet cell starvation, hypoxia and death. The mechanism of PFO formation is still not elucidated fully and studies using a pig model have tried to understand the host immune response to empty alginate microcapsules. In this review, the varied strategies to overcome or reduce PFO are discussed, including alginate purification, altering microcapsule geometry, modifying alginate chemical composition, co-encapsulation with immunomodulatory cells, administration of pharmacological agents, and alternative transplantation sites. Nanoencapsulation technologies, such as conformal and layer-by-layer coating technologies, as well as nanofiber, thin-film nanoporous devices, and silicone based NanoGland devices are also addressed. Finally, this review outlines recent progress in imaging technologies to track encapsulated cells, as well as promising perspectives concerning the production of insulin-producing cells from stem cells for encapsulation.

Noninvasive Tracking of Encapsulated Insulin Producing Cells Labelled with Magnetic Microspheres by Magnetic Resonance Imaging.

Microencapsulated islets are usually injected free-floating into the peritoneal cavity, so the position of the grafts remains elusive after transplantation. This study aims to assess magnetic resonance imaging (MRI) as a noninvasive means to track microencapsulated insulin producing cells following transplantation. Encapsulated insulin producing cells (MIN6 and human islets) were labelled with magnetic microspheres (MM), assessed for viability and insulin secretion, and imaged in vitro using a clinical grade 3 T MRI and in vivo using both clinical grade 3 T and research grade 11.7 T MRI. Fluorescent imaging demonstrated the uptake of MM by both MIN6 and human islets with no changes in cell morphology and viability. MM labelling did not affect the glucose responsiveness of encapsulated MIN6 and islets in vitro. In vivo encapsulated MM-labelled MIN6 normalized sugar levels when transplanted into diabetic mice. In vitro MRI demonstrated that single microcapsules as well as clusters of encapsulated MM-labelled cells could be visualised clearly in agarose gel phantoms. In vivo encapsulated MM-labelled MIN6 could be visualised more clearly within the peritoneal cavity as discrete hypointensities using the high power 11.7 T but not the clinical grade 3 T MRI. This study demonstrates a method to noninvasively track encapsulated insulin producing cells by MM labelling and MRI.

Coencapsulation of Target Effector Cells With Mesenchymal Stem Cells Reduces Pericapsular Fibrosis and Improves Graft Survival in a Xenotransplanted Animal Model.

Pericapsular fibrotic overgrowth (PFO) is a problem that thwarts full implementation of cellular replacement therapies involving encapsulation in an immunoprotective material, such as for the treatment of diabetes. Mesenchymal stem cells (MSCs) have inherent anti-inflammatory properties. We postulated that coencapsulation of MSCs with the target cells would reduce PFO. A hepatoinsulinoma cell line (HUH7) was used to model human target cells and was coencapsulated with either human or mouse MSCs at different ratios in alginate microcapsules. Viability of encapsulated cells was assessed in vitro and xenografted either intraperitoneally or subcutaneously into C57BL/6 mice. Graft retrieval was performed at 3 weeks posttransplantation and assessed for PFO. Coencapsulation of human MSCs (hMSCs) or mouse MSCs (mMSCs) with HUH7 at different ratios did not alter cell viability in vitro. In vivo data from intraperitoneal infusions showed that PFO for HUH7 cells coencapsulated with hMSCs and mMSCs in a ratio of 1:1 was significantly reduced by ∼30% and ∼35%, respectively, compared to HUH7 encapsulated alone. PFO for HUH7 cells was reduced by ∼51% when the ratio of mMSC/HUH7 was increased to 2:1. Implanting the microcapsules subcutaneously rather than intraperitoneally substantially reduced PFO in all treatment groups, which was most significant in the mMSC/HUH7 2:1 group with a ∼53% reduction in PFO compared with HUH7 alone. Despite the reduced PFO reaction to the individual microcapsules implanted subcutaneously, all microcapsule treatment groups were contained in a vascularized fibrotic pouch at 3 weeks. The presence of MSCs in microcapsules retrieved from these fibrotic pouches improved graft survival with significantly higher cell viabilities of 83.1 ± 0.6% and 79.1 ± 0.8% seen with microcapsules containing mMSC/HUH7 at 2:1 and 1:1 ratios, respectively, compared to HUH7 alone (51.5 ± 0.7%) transplanted subcutaneously. This study showed that coencapsulation of MSCs with target cells has a dose-dependent effect on reducing PFO and improving graft survival when implanted either intraperitoneally or subcutaneously in a stringent xenotransplantation setting.

Clinical use of GLP-1 agonists and DPP4 inhibitors.

Type 2 diabetes is a growing problem, with 387 million people currently affected, and 592 million by 2035. Whilst diet and exercise are the corner stones of treatment, oral hypoglycaemic agents are often needed to achieve glycaemic control, thereby reducing the chance of long term diabetic complications. Biguanides and sulfonylureas have been the standard tablets used for this disorder, until 2005-7 when glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP4) inhibitors became available. Their major advantage over sulfonylureas is that they are weight lowering or weight neutral, and have a very low incidence of hypoglycaemia. GLP-1 agonists are injectables, whereas the DPP4 inhibitors are administered orally. Both agents are best used in combination with other hypoglycaemic medication, especially metformin and sodium glucose co-transporter 2 (SGLT2) inhibtors. Usage is increasing, being roughly equal to that of sulfonylureas, but less than that of metformin. Side effects appear to be minimal.

Scaffolds for islets and stem cells differentiated into insulin-secreting cells.

Embryonic/pluripotent stem cells offer the possibility of an unlimited source of cells to be differentiated into beta cells. This requires differentiating the stem cells into pancreatic progenitors by tissue culture, and then transplanting into recipients for the final stages of development into mature beta-cells. Exposing embryonic stem cells seeded onto laminin coated PLGA scaffolds to biochemical cues resulted in enhanced expression of definitive endoderm markers compared to those differentiated on 2D monolayers. The production of tissue specific cells from stem cells can be scaled up using bioreactor cultures. To apply human stem cell derived islet progenitors in a clinical setting, one must first overcome the problem of immune rejection. Immuno-isolating the cells using microencapsulation provides one possible solution. Coating scaffolds with an anti-inflammatory agent could be an effective means of reducing the inflammatory process that results in pericapsular fibrosis and necrosis of the encapsulated cells. This review summarizes the above issues and describes how 3D scaffolds seeded with stem cells and/or pancreatic progenitors may provide a benefit to achieving normalization of blood glucose levels.

Beneficial effects of coating alginate microcapsules with macromolecular heparin conjugates-in vitro and in vivo study.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated pancreatic islets. Modification of the microcapsule membrane aimed at preventing PFO should improve graft survival. This study investigated the effect of macromolecular Corline Heparin Conjugate (CHC) binding on intrinsic properties of alginate microcapsules and assessed the anti-fibrotic potential of this strategy both in vitro and in vivo. CHC was bound to alginate microcapsules using a layer-by-layer approach incorporating avidin. CHC binding to alginate microcapsule was visualized by confocal microscopy. Effects of CHC binding on microcapsule size, strength, and permeability were assessed, and the anti-clotting activity of bound CHC was determined by coagulation assay. Effect of CHC binding on the viability of encapsulated human islets was assessed in vitro, and their ability to function was assessed both in vitro and in vivo in diabetic immunodeficient mice. The potential of bound CHC to reduce PFO was assessed in vivo in different rat transplantation models. Confocal microscopy demonstrated a uniform coating of CHC onto the surface of microcapsules. CHC binding affected neither size nor permeability but significantly increased the tensile strength of alginate microcapsules by ~1.3-fold. The bound CHC molecules were stable and retained their anti-clotting activity for 3 weeks in culture. CHC binding affected neither viability nor function of the encapsulated human islets in vitro. In vivo CHC binding did not compromise islet function, and diabetes was reversed in all recipients with mice exhibiting lower blood glucose levels similar to controls in oral glucose tolerance tests. CHC binding was beneficial and significantly reduced PFO in both syngeneic and allogeneic rat transplantation models by ~65% and ~43%, respectively. In conclusion, our results show a new method to successfully coat CHC on alginate microcapsules and demonstrate its beneficial effect in increasing capsule strength and reduce PFO. This strategy has the potential to improve graft survival of encapsulated human islets.

Hepatocyte-like cells derived from human amniotic epithelial cells can be encapsulated without loss of viability or function in vitro.

Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro.