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Engineering the plasma membrane of pancreatic beta cells for a cell-based treatment in T1DM
Introduction: Type 1 diabetes mellitus (T1DM) results from a specific immune-mediated destruction of pancreatic β cells that leads to the complete loss of insulin production. Since the discovery of insulin in 1921, T1DM has been effectively treated, however, exogenous insulin replacement therapy still does not mimic the physiological pancreatic insulin secretion pattern, which results in the occurrence of life threating hypoglycaemic episodes and in the increase of incidence of macro and microvascular complications. Currently, T1DM treatment is shifting towards pancreatic β cell replacement for the restoration of responsive insulin secretion to blood glucose variations. Although the infusion of isolated islets through the portal vein of the liver is possible, the shortage of donors and the dangers of pharmacological immunosuppression makes it an unlikely approach for T1DM. Research is now being carried out to develop novel cell-based therapies for T1DM in order to isolate cells from the immune system and avoid the use of immunosuppression drugs, with the ambition of restoring insulin competence in patients with T1DM.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
Aims: In this landscape, we propose to immunoprotect pancreatic β cells by bioengineering their plasma membrane using PEG-lipids. Methods: Dipalmitoyl-glycerol-phosphatidyl ethanolamine (DPPE) 20 mg and maleimide-polyethylene glycol-N-hydroxysuccinimide (mal-PEG-NHS) 180 mg were dissolved in chloroform and stirred for 24 hours at room temperature to form mal-PEG-lipid. The product was purified by precipitation in diethyl ether followed by centrifugation (600 RPM), where the pellet is obtained after evaporation. Mal-PEG-Lipids (20 mg) were labelled using 3.1 mg of fluorescein-5-isothiocyanate (FITC). Beta-TC-6 cells derived from pancreatic tumour (insulinoma) had their surface membrane modified by the insertion of FITC-mal-PEG-lipids. A suspension of cells (5x105) were incubated with 1,000 μM of FITC-mal-PEG-lipids for 120 minutes at 37°C. Cells were centrifuged (180 G) and washed with PBS twice. Cell membrane modification was confirmed using confocal microscopy. The integrity of the plasma membrane after the modification was assessed using annexin V labelled with Alexa Fluor 568. Results and Discussion: Palmitoyl was selected to be inserted into the lipid bilayer by hydrophobic adsorption because saturated fatty acids are more stable in the plasma membrane than unsaturated fatty acids; long fatty acids (above 13 carbons) also show to be more stable in the plasma membrane than short fatty acids and do not change the membrane fluidity like very long fatty acids (above 21 carbons); and palmitoyl is a native fatty acid of plasma membranes. Mal-PEG was also preferred to modify the plasma membrane over biotin-streptavidin because it is less immunogenic than the former. Surface engineering of pancreatic β cells was possible and showed no harmful effects to the cells.
Considerações Finais/Final considerations/Consideraciones finales
Final considerations: Next steps involve the additional coating of pancreatic β cells using hyaluronic acid derivates that are attached to the plasma membrane by reacting with the maleimide portion of the PEG-lipids. Coating stability and diffusion characteristics will be assessed to validate the coating as a barrier for the immune system while allowing the diffusion and release of glucose and insulin, respectively.
Palavras-chave/Key words/Palabras clave
Type 1 Diabetes Mellitus, Pancreatic Beta Cells, Immunoprotection, Surface Engineering
FERNANDA ZAMBONI, Maurice N. Collins