title: Optimising islet transplantation therapy for the treatment of type 1 diabetes by targeting the liver niche with Etanercept-loaded microparticles to promote the long term function of islets
Creators: name: Walker, Sophie Louise
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: subject: Type 1 diabetes
subject: islets
subject: insulin
subject: islet transplant
subject: Etanercept
subject: mouse model
subject: microparticles

description: Type 1 diabetes is characterised by the autoimmune destruction of β-cells within the islets of Langerhans, which are present within the pancreas. This results in dysregulation of blood glucose levels, and results in hyperglycaemia and diabetic ketoacidosis. Current treatment options include the administration of exogenous insulin, which can result in hypoglycaemia with typical initial symptoms being dizziness, sweating and mumbled speech but can quickly lead to comas and death if not treated. Some people with type 1 diabetes do not have hypoglycaemic awareness and can experience hypoglycaemia without being aware of the symptoms mentioned above. Islet transplantation into the liver is offered to restore hypoglycaemic awareness in some patients and importantly improve quality of life. However, islet transplantation is associated with a number of problems, including: a scarcity of the islet resources; 2-3 pancreases of islets being needed per transplant due to the high level of islet loss within the first 72 hours post-transplant because of inflammation and the islets being avascular upon transplantation; and the requirement for patients to be on immunosuppression for the lifetime of the graft. Etanercept is an anti-inflammatory agent targeting tumour necrosis factor alpha (TNFα), which is administered subcutaneously during islet transplantation along with immunosuppressive agents and at high concentrations can cause islet toxicity. Our aim was to improve islet transplant outcomes through the reduction of inflammation in the liver following islet transplantation by using Etanercept-loaded microparticles, which specifically target the liver. Using the liver-specific microparticles is hypothesised to more efficaciously reduce inflammation in the liver compared with subcutaneous doses of the agent at a fraction of the dose in order to improve islet survival and overall islet health. Galactosylation of poly(lactic-co-glycolic acid) (PLGA) microparticles, formed via the double emulsion method of microparticle manufacture, resulted in microparticles being formed to bind specifically to asialoglycoprotein receptors (ASGPRs) which are solely expressed in the liver, this has been established in previous studies, however a gap in knowledge in the 10-26 µm microparticle range has been noted. In this study, 13 µm and 20 µm diameter PLGA only and galactosylated-PLGA microparticles were tested at 0.1 mg and 1 mg doses. The preferential formulation and dose to achieve liver specific microparticle dosing with minimal liver necrosis is 0.1 mg of 13 µm galactosylated-PLGA microparticles. A burst of encapsulated drug and then sustained release over 14 days was observed in vitro, which is a preferential release profile due to the highest amount of islet loss being observed in the first 72 hours post-transplant. In vitro glucose stimulated insulin secretion (GSIS) studies of mouse islets showed a statistically significant increase in stimulation index (SI), normalised to protein content, when islets were subjected to an overnight incubation with 0.1 mg of Etanercept microparticles prior to the GSIS study, compared to when islets were incubated alone. A burst and then sustained release of Etanercept was also observed in C57Bl/6 mice over a 3-day study period, following a similar trend to that seen in vitro. Currently, open laparotomy surgical techniques are routinely used for injection into the hepatic portal vein (HPV) in mice. We sought to determine whether ultrasound-guided HPV injection could be used as a methodological refinement, which would better emulate clinical HPV injections. Open laparotomy HPV injection was seen to be superior, with a higher number of fluorescent-PLGA microparticles being delivered to the liver, lung and spleen tissues, showing that the microparticles had successfully entered the systemic circulation – a main outcome of this specific study to show successful injection. Very few microparticles were seen in liver tissue following ultrasound-guided injection, with no microparticles residing in the lung and spleen tissues, suggesting that the injections had been unsuccessful, and so open laparotomy approaches should be continued to be used. Syngeneic co-transplant studies of mouse islets with Etanercept-loaded galactosylated-PLGA microparticles were performed in C57Bl/6 mice rendered diabetic using Streptozotocin. Initial co-transplant studies with 350 islets and 0.1 mg of 13 µm galactosylated-PLGA microparticles showed no resolution of diabetes at 6 weeks post-transplant, however a statistically significant increase in terminal serum insulin content in the group receiving islets with Etanercept microparticles was observed, compared to when only islets were injected. Insulin staining was observed in liver tissues, however no statistically significant changes were observed in insulin staining area, liver function tests, or CD3 inflammatory staining between the three study groups (islets alone, or with the addition of blank or Etanercept-loaded microparticles). Subsequent studies using 650 islets co-transplanted with Etanercept microparticles showed no statistically significant changes in any of the above-mentioned end-point measurements between the two study groups (islets alone, or with the addition of Etanercept-loaded microparticles). In conclusion, galactosylation of 13 µm PLGA microparticles results in liver-specific dosing in mice, with burst and then sustained release of Etanercept being achieved. Open laparotomy surgical HPV injections should continue to be used in pre-clinical studies of islet transplantation, until a refined alternative to better emulate clinical islet transplant can be used. Promising results were seen in in vitro and in initial in vivo studies of mouse islets with Etanercept microparticles, however this was not recapitulated in subsequent in vivo studies with higher mouse islet numbers being transplanted. Refinement and continuation of studies to examine Etanercept-loaded microparticles is needed to determine if their presence improves islet transplant to a level which would be clinically beneficial; alternative or combination loading of microparticles may have more clinical benefit which should be further investigated.