In Vitro Evaluation of Necrostatin-1 Microparticles for Limiting Stress-induced Cell Death and Preserving Islet...

Islet transplantation offers a promising therapy for type 1 diabetes, but early β-cell loss due to stress-induced cell death limits graft survival and function. Although apoptosis has been extensively studied, necroptosis, a regulated form of necrotic cell death, remains an underexplored contributor to β-cell dysfunction. Herein, we evaluated sustained necrostatin-1 (Nec-1) delivery via poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) to protect islets from stress-induced damage in vitro.

Nec-1-loaded PLGA MPs were synthesized by single-emulsion solvent evaporation and characterized by scanning electron microscopy and high-performance liquid chromatography for size, morphology, and drug encapsulation. Mouse islets were coincubated with Nec-1 or empty MPs for 24 h, followed by induction of endoplasmic reticulum stress with thapsigargin. Glucose-stimulated insulin secretion and cytokine release were quantified to assess β-cell function and inflammatory response.

Nec-1 MPs were spherical with a median diameter of 10.34 μm (interquartile range [IQR], 6.55-15.17 μm) and an encapsulation efficiency of 36.2% (IQR, 31.1%-40.0%). Release studies demonstrated an initial burst of 24.8% (IQR, 21.8%-30.3%) by day 3, followed by sustained delivery up to 53.6% (IQR, 52.3%-57.4%) by day 14, with minimal impact on media pH. Under thapsigargin-induced stress, Nec-1 MP-treated mouse islets maintained glucose-stimulated insulin secretion, with insulin release increasing from 2.63% (IQR, 1.42%-5.74%) to 8.63% (IQR, 7.97%-14.42%, P = 0.0006) compared with 3.36% (IQR, 0.53%-9.08%) in stressed controls (P = 0.0117). The Stimulation Index was similarly preserved (3.23; IQR, 2.51-4.77 versus 1.18; IQR, 0.65-1.62; P = 0.0002). Nec-1 MPs partially attenuated IL-12p70 secretion (103.3 pg/mL; IQR, 67.7-162.4 versus 217.1 pg/mL; IQR, 86.2-367.6; P = 0.0381) while maintaining low levels of other proinflammatory cytokines.

Nec-1-loaded PLGA MPs provide sustained, localized protection of β-cells from necroptotic and inflammatory stress in vitro. Given the sustained and targeted drug delivery, these findings provide a foundation for future in vivo translation to provide graft-localized therapeutic intervention and improve islet survival, engraftment, and long-term function.