Nano-scale encapsulation enhances allograft survival and function of islets transplanted in a mouse model of diabetes
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The success of islet transplantation as a treatment for type 1 diabetes is currently hampered by post-transplantation loss of functional islets through adverse immune and non-immune reactions. We aimed to test whether early islet loss can be limited and transplant survival improved by the application of conformal nano-coating layers to islets.
Our novel coating protocol used alternate layers of phosphorylcholine-derived polysaccharides (chitosan or chondroitin-4-sulphate) and alginate as coating materials, with the binding based on electrostatic complexation. The in vitro function of encapsulated mouse islets was studied by analysing islet secretory function and cell viability. The in vivo function was evaluated using syngeneic and allogeneic transplantation in the streptozotocin-induced mouse model of diabetes.
Nano-scale encapsulated islets retained appropriate islet secretory function in vitro and were less susceptible to complement- and cytokine-induced apoptosis than non-encapsulated control islets. In in vivo experiments using a syngeneic mouse transplantation model, no deleterious responses to the coatings were observed in host animals, and the encapsulated islet grafts were effective in reversing hyperglycaemia. Allo-transplantation of the nano-coated islets resulted in preserved islet function post-implantation in five of seven mice throughout the 1 month monitoring period.
Nano-scale encapsulation offers localised immune protection for implanted islets, and may be able to limit early allograft loss and extend survival of transplanted islets. This versatile coating scheme has the potential to be integrated with tolerance induction mechanisms, thereby achieving long-term success in islet transplantation.
KeywordsAllogeneic transplantation Graft survival Immunoisolation Islet transplantation Nano-coating Nano-scale encapsulation Type 1 diabetes
Hanks’ buffered salt solution
Transmission electron microscopy
Islet transplantation is arguably one of the most important conceptual advances in the treatment of type 1 diabetes and has the potential to cure the disease . A major barrier to successful transplantation is early post-transplantation depletion of functional islets in response to activated complement and coagulation systems, and to a chronic inflammatory and immunogenic environment [2, 3, 4, 5]. This has obvious detrimental effects on the outcome of individual grafts and further exacerbates the scarcity of donor tissue. Moreover, allogeneic transplantation requires pharmacological suppression of the host immune system to circumvent graft rejection, while current immunosuppressive regimens are likely to contribute to the early loss of engrafted islets . A strategy that conceals implanted islets from the host immune system may help to minimise or even prevent the post-transplantation loss of islet mass and function by immune rejection. Thus physical isolation of islets from the host environment by encapsulation, while maintaining islet responsiveness to metabolic changes, could be an effective means of improving islet survival and function after transplantation.
Islet encapsulation strategies to date have mainly focused on macrocapsules (encapsulation of the whole islet graft) and microcapsules (encapsulation of individual islets) . Previous studies in animal models [8, 9] and in human participants [10, 11] have demonstrated that physical isolation of islets from the host immune system by, for example, alginate microencapsulation is effective in preventing beta cell loss and in maintaining long-term secretory function in transplanted allogeneic and xenogeneic islets without systemic immunosuppression [12, 13, 14, 15, 16]. Although conferring the immunological advantages predicted from graft/host isolation, microcapsules have some significant drawbacks. Thus the relatively large volume of a typical alginate capsule (diameter ∼500–800 μm) compared with a typical islet (∼150 μm) results in a greatly increased diffusion length, which can lead to impaired diffusion of oxygen and nutrients to the islet, with consequent hypoxic cell death or malfunction. A more immediate consequence of the size of microcapsules is the choice of anatomical location for the graft material, with most experimental studies focussing on intraperitoneal or subcutaneous compartments due to their large capacity . In contrast, intraportal infusion is the current site of choice for clinical programmes , with the transplanted islets lodging in the hepatic microcirculation. An encapsulation technology designed for application to current clinical transplantation of human islets must therefore be compatible with intraportal delivery to the hepatic capillary bed, excluding microcapsules or macrocapsules on the basis of their size.
Conformal nano-coating avoids these problems by generating a biocompatible nanometre-scale isolating layer close to the cell surface, thus reducing barriers to diffusion, while ensuring that the encapsulated islets can be implanted into any site suitable for non-encapsulated islets . The challenge with conformal nano-coating is to nano-engineer an efficient and lasting immune-protective layer that covers islets completely or nearly completely, and is biocompatible with the recipient.
Several methods of conformal coating have been developed recently, including covalent surface attachment of polyethylene glycol (PEG), known as ‘PEGylation’ , and layer-by-layer encapsulation [19, 20, 21, 22, 23, 24, 25, 26, 27, 28]. Nano-layers with different combinations of components, including streptavidin and biotin–PEG derivatives [19, 24], complement receptor 1 and heparin , and PEG–lipid and poly(vinyl alcohol) [26, 27], have been previously researched as islet surface coatings. Most of these attempts at islet encapsulation have focused on development of the method, with relatively little emphasis on in vivo evaluation of the technology, and particularly on the maintenance of functional beta cells in an allogeneic environment. Teramura and Iwata demonstrated that coating of islet surfaces with PEG-lipid and urokinase could effectively protect against blood-mediated inflammatory reactions in a syngeneic mouse model of transplantation . However, a recent study using conformal coating to encapsulate islets via layer-by-layer deposition of poly(l-lysine)-g-PEG-(biotin) and streptavidin failed to show any improvement in the survival and function of the implanted allo-islets . Effective immunoprotection using nano-coating thus remains a challenge in islet transplantation.
A more detailed description of research design and methods, including regents and materials, is available in the electronic supplementary material (ESM Methods).
For syngeneic transplantation, male C57BL/6 mice aged 8 weeks and weighing 20–25 g (Charles River, Margate, UK) were used as donors and recipients of grafts. For allogeneic transplantation, male C57BL/6 mice were used as islet graft recipients and Balb/c mice as tissue donors. Recipient mice were made diabetic by a single i.p. streptozotocin injection (180 mg/kg; Sigma-Aldrich, Poole, UK) 5–6 days prior to transplantation and those with a non-fasting blood glucose concentration of ≥20 mmol/l were used as recipients. All animal procedures were approved by our institution’s Ethics Committee and carried out under licence, in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986.
Islets were isolated from mice by injecting collagenase (type XI, 1 mg/ml; Sigma-Aldrich) into the pancreas via the common bile duct, followed by digestion for 10 min at 37°C. The islets were purified by centrifugation (3,500 rpm, 25 min; Universal 320R, Hettich Zentrifugen, Tuttingen, Germany) in a density gradient (Histopaque-1077; Sigma-Aldrich). Before being used for encapsulation, the purified islets were incubated for 16 h in RPMI-1640 medium (Sigma-Aldrich) that was supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin.
Nano-coating of islets
The working solutions of the polysaccharides, including PC-chitosan, alginate and PC-modified chondroitin-4-sulphate (1 mg/ml; Fig. 1), were prepared by dissolving the solids in Hanks’ buffered salt solution (HBSS; PAA Laboratories, Pasching, Austria), which was pH-adjusted to 6.9–7.0 and supplemented by 2 mmol/l CaCl2. Positively charged chitosan-PC and negatively charged alginate (unmodified) were alternately deposited into a multilayer film on individual islets in suspension in a 1.5 ml Eppendorf tube. Cationic chitosan-PC (400 μl) was added first to a suspension of the islets (pre-washed with HBSS) to form a seeding layer. After 5 min deposition time, two intermediate washings with the buffer were made using gravity sedimentation for 1 to 2 min to remove any excess, unadsorbed chitosan-PC. Subsequently, anionic alginate (400 μl) was adsorbed in the same manner. This process was repeated n times per batch of islets according to the following layering scheme: islets/(chitosan-PC–alginate layers) n , where n represents the number of bilayers. Finally, a PC-modified chondroitin-4-sulphate (400 μl) layer was added as the outermost layer in the same manner as for alginate. Some loss of islets was incurred, totalling <10% for a typical eight-layer coating process. All encapsulation solutions were filtered through a sterile 0.2 μm membrane filter cartridge. All coating, washing and sample solutions were kept on ice during the coating process.
Dynamic insulin secretion from the encapsulated islets
Encapsulated islets were incubated for 16 h (37°C) before perifusion. The rate and patterns of in vitro insulin secretion from encapsulated and control islets were assessed using a temperature-controlled (37°C) multi-channel perifusion system, as described previously . Briefly, 40 islets were loaded on to nylon filters in Swinnex filter holders (Millipore, Cork, Ireland) and perifused with a bicarbonate-buffered physiological salt solution (Gey & Gey buffer, made in house) supplemented by 2 mmol/l CaCl2, 0.5 mg/ml bovine serum albumin and a concentration of glucose as indicated below. Fractions were collected every 2 min during (1) a 10 min perifusion period with buffer containing 2 mmol/l glucose, (2) a 20 min perifusion with 20 mmol/l glucose and (3) an additional 20 min perifusion with 2 mmol/l glucose. Insulin content was assessed by radioimmunoassay .
Transplantation of encapsulated islets in diabetic mice
Mice were anaesthetised by inhalation of isoflurane and transplanted with 300 islets under the kidney capsule, according to a procedure reported previously . Briefly, a lumbar incision was made, the kidney exposed and an incision made in the capsule. Encapsulated and control islets that had been centrifuged into pellets in PE50 polyethylene tubing (Becton Dickinson, Franklin Lakes, NJ, USA) were placed underneath the kidney capsule using a Hamilton syringe (Fisher, Two Rivers, WI, USA). All islets were transplanted with a delay of no more than 2 h after encapsulation.
In vivo function of islet graft
The body weight and blood glucose concentrations of recipient mice were monitored every 1–2 days. Reversal of hyperglycaemia was defined as non-fasting blood glucose concentrations ≤11.1 mmol/l on at least two consecutive readings. In cured animals, we assessed the in vivo function of the transplanted islets by an intraperitoneal glucose tolerance test at 1 month after transplantation. Weight-matched, non-diabetic, non-transplanted male C57BL/6 mice were used as controls. Fasting blood glucose concentrations were measured prior to an i.p. injection of 2 g/kg of glucose dissolved in saline solution and then after 15, 30, 60, 90 and 120 min. The islet graft-bearing kidneys were removed 1 or 2 days later to assess whether graft removal would result in a reversion to hyperglycaemia. Other mice in which the graft was rejected in less than 28 days were killed and the graft-bearing kidney removed for histological analysis.
Detailed descriptions of histological and immunohistological analysis of the graft-bearing kidneys is available in the ESM Methods.
Independent t tests were used to test for significant difference between individual groups of the cytokine assay results and glucose tolerance data. Values of p < 0.05 were considered significant.
Deposition of coating layers on islets
The deposition of the non-labelled polysaccharide multilayers on the individual islets was further confirmed by high-resolution transmission electron microscopy (TEM) imaging of the encapsulated islets (Fig. 3d; ESM Methods). The ultrastructural image showed an intact coating consisting of nano-layers of approximately 80 nm thickness that covered the outer surface of cells on the islet periphery. The nano-layer was not found in the extracellular space between the islet cells. Insulin-secreting vesicles (dense-core granules) were seen by TEM to be aligned along the plasma membrane ready for exocytosis, suggesting that beta cells were healthy and unaffected by the coating.
In vitro cytotoxicity exerted by the coating materials
In addition, no elevated level of apoptosis in the nano-coated islets was detected after 48 h in culture compared with non-encapsulated islets, as shown in Fig. 4c (see ESM Methods). The rate of islet cell apoptosis (determined as caspase-3/7 activity) in control islets was increased by approximately 620% following exposure to combined cytokines (IL-1β 1.7 ng/ml, TNF-α 1.7 ng/ml and IFN-γ 3 ng/ml), but this was significantly less in the nano-coated islets. The negative coating layer with alginate was more potent in inhibiting cytokine-induced cell damage than chondroitin-4-sulphate-PC. Nano-coated islets were also protected against apoptosis mediated by complement (50% rabbit serum; Fig. 4d). Chondroitin-4-sulphate-PC, an anti-coagulatory molecule [31, 32], was found to provide a better protective effect than alginate. Based on the findings above, the coating scheme adopted for our in vivo experiments used chitosan-PC and alginate in repeated layers, with the last layer being chondroitin-4-sulphate-PC.
Assessment of the in vitro function of nano-scale encapsulated islets
In vivo assessment of nano-scale encapsulated islet function: syngeneic transplantation
In vivo assessment of nano-scale encapsulated islet function: allogeneic transplantation
Protection from immune assault was more successful in a further study, in which we implanted islets coated with eight layers in the allogeneic model. In this study, the graft material was retrieved after about 1 month for histological analysis. Nano-scale encapsulated islets maintained normoglycaemia for 28–37 days (Fig. 7b) in five of seven mice. Reversion to hyperglycaemia for mice receiving encapsulated islet transplants was caused by graft-bearing kidney removal. Two mice receiving the encapsulated islets reverted to hyperglycaemia in less than 28 days due to rejection.
To determine whether transplanted islets secrete insulin and maintain glycaemic control in response to increases in blood glucose levels, we carried out intraperitoneal glucose tolerance tests at 4 weeks post-transplantation in mice that had remained normoglycaemic. Figure 7c shows that the change in glucose level was similar to that of non-diabetic mice. Histological analysis of the recovered grafts showed significant amounts of insulin-positive tissue (Fig. 7e) and no evidence of infiltration of T cells into the islets, although a few macrophages were detected (ESM Fig. 3).
We describe here a new protocol for nano-scale encapsulation of pancreatic islet cells and for the first time demonstrate in an animal model improved post-transplant survival of such coated islet cells in comparison with uncoated islets.
Alternate layers of PC-grafted polysaccharides (chitosan and chondroitin-4-sulphate) were used with alginate as the coating materials, with the binding being based on electrostatic complexation. Multilayer film formation is possible because of charge reversal on the film surface after each adsorption step. The polysaccharides we used contain at least one charge in each monosaccharide unit, which serves as the binding site, offering hundreds of binding sites for each polysaccharide molecule, thus providing a strong affinity to the charged cellular surfaces. The rationale for using these polymers in the layer architecture was based on our in vitro studies, which identified potential complement- and inflammation-inhibitory activities of these polysaccharides (Fig. 3c, d). Any success of islet encapsulation in extending graft survival in this study is likely to be dependent to a large part on the ability of the nano-coating to control the local transplant microenvironment and restrict immune cell infiltration, essentially blocking a variety of protein–protein interactions involved in complement (molecular mass 500 kDa), macrophage and T cell activation. In this regard, we introduced the protein-repelling zwitterionic PC modification (Fig. 1) in the coating constructs to minimise interactions between islets and the environment. The PC moiety, which is a component of plasma cell membranes, confers hydrophilicity, haemocompatibility and resistance to non-specific protein absorption, thus inhibiting the development of fibrosis, supporting endothelial cell growth  and also carrying anti-coagulatory properties [37, 38], all of which should enhance islet survival in vivo and encourage host integration. In addition, the 40% amine group content of the PC modification for chitosan increased the solubility of the polysaccharide up to about 2 mg/ml under physiological pH conditions (pH ∼ 7.0).
Using the layer-by-layer deposition technique, a defined multilayer coating containing PC modification could be immobilised on the islet cell surfaces (ESM Fig. 5). The thickness (80 nm) of a typical eight-layer coating, as measured by TEM, is consistent with that previously reported with similar polymer sizes (200 nm with 20 layers) . The addition of a polymer, chondroitin-4-sulphate, that carried a strongly ionic sulphate group into the complexation pair confers anti-coagulatory properties to the coating. It also contributes greatly to the stability of the layered nanofilm , making it a very durable coating even under harsh physiological conditions.
The nano-scale encapsulated mouse islets were found to preserve appropriate islet secretory function and survival in vitro. This indicates that the multilayers of the polysaccharides were deposited non-covalently on to the cell surfaces without perturbing cellular physiology or compromising cell survival. Pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-1β, are major products of activated effective T cells and macrophages, and are known to be damaging to pancreatic islets via apoptosis induction . In this study, we found that the nano-coating also rendered the islets less susceptible to cytokine- and complement-induced apoptosis (Fig. 4c, d). In addition, we found that the PC-displayed nano-coating effectively inhibited specific adsorption of large molecules of the immune systems (IgG, 150 kDa) on to the islet cell surfaces (ESM Fig. 6), indicating the effectiveness of the non-fouling PC-modification of the coating materials.
We used the syngeneic transplantation model to assess the in vivo secretory functionality of nano-scale encapsulated mouse islets and their ability to reduce hyperglycaemia and maintain normoglycaemia thereafter. These studies were used to avoid any influences of graft-versus-host immune rejection on islet function, while focusing on how the coating layers could impact on the immediate inflammatory reaction. Our in vivo tests using the syngeneic mouse transplantation model showed no deleterious responses from host animals to the coating materials, suggesting the materials used for encapsulation are non-toxic. Allo-transplantation studies in present work have shown that tailored encapsulating layers could optimise islet function post-implantation, allowing a degree of protection against inflammation and immune rejection in the majority of the studies. In the current study, nano-scale encapsulated islets were responsive to a hyperglycaemic environment, secreted appropriate amounts of insulin to restore normoglycaemia and survived for an extended period in vivo.
The failure of encapsulation to prevent rejection in two of the seven animals studied is of note and may indicate incomplete coating before transplantation or degradation at some time after transplantation. Future studies will need to test whether the robustness or completeness of the coating can be improved.
Since the purpose of our study was to prevent or reduce early-stage islet loss, we considered that a 4–5 week post-transplant observation period was appropriate. The rejection in controls normally occurs at 10–14 days (Fig. 7a), so the chosen study period showed that graft survival was twice as long. We have to look at grafts when the animals are normoglycaemic to determine whether there are any signs of graft destruction or whether the graft has a normal histological appearance. A long-term study of the function and graft survival with this multilayer coating scheme has not yet been performed. Nevertheless, unlike the arguably more expected durability of alginate microcapsules [15, 42], the nano-layers used in this study, while being non-biodegradable, are not expected or intended to last for many months under in vivo conditions. Rather, we propose that nano-scale encapsulation is an alternative technology that may promote engraftment by allowing intraportal administration and by limiting early islet loss caused by exposure to an inflammatory environment. In fact, our in vitro cytokine exposure study (Fig. 3c, d) confirmed that the encapsulation layers were protective against the cytotoxic effects of cytokines and complement proteins, as assessed by a reduction of apoptosis. Thus by the integration of graft tolerance induction mechanisms, the encapsulated environment may promote the development of regulatory T cells, which inhibit unintended host immune system activation, thereby achieving permanent survival of the transplanted islets [43, 44].
Studies are now needed to test whether modifications of the nano-layers will enhance engraftment and prevent rejection of islets in the longer term. This may involve: (1) incorporating bioactive molecules such as complement and coagulation inhibitors into the layers to prevent islet loss caused by the instant blood-mediated inflammatory reaction; (2) incorporating anti-inflammatory agents to reduce localised inflammation and fibrosis; or (3) incorporating natural and/or artificial extracellular matrices to provide optimal cell functioning after engraftment. Since there is little or no increase in size and volume of the islets after encapsulation, we expect this protocol will be potentially suitable for hepatic implantation via intraportal infusion. Such an option now needs to undergo experimental testing.
The authors acknowledge the Engineering and Physical Sciences Research Council (EPSRC) (UK) Science and Innovation award (EP/D062861/1) for generous grant supports.
ZlZ contributed to the conception and design of the study, performed the experiments and wrote the manuscript. AK contributed to the interpretation of in vivo data. AJFK contributed to the conception and design of in vivo experiments. PMJ contributed to the conception and design of the study, and reviewed the manuscript. JCP contributed to the conception and design and reviewed/edited the manuscript. All authors have read and given critical input during preparation of the manuscript and all have approved the final version.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.