Degradable Nanoparticles Restore Lysosomal pH and Autophagic Flux in Lipotoxic Pancreatic Beta Cells

Abstract

between autophagosome and a sufficiently acidic lysosome (Figure 1). In the insulin secreting pancreatic beta cells (β-cells), chronic exposure to high levels of fatty acids results in lipotoxicity [7,8] and leads to disruption of autophagy concomitant with compromised lysosomal acidity. This autophagic dysfunction affords an increased accumulation of damaged organelles and proteins in the cells, thereby exacerbating the decline in cellular functions which leads to progressive diseases such as Type II diabetes (T2D). [7][8][9][10][11][12][13][14][15] Therefore, addressing the role of impaired acidification in lipotoxic pancreatic β-cells is important for identifying its downstream consequences and for studying the potential benefits of restored acidification on cellular function and autophagy. Given that nanoparticles (NPs) readily accumulate in lysosomes upon exposure to cells, nanoparticle-based strategies that alter the local intracellular lysosomal pH are being explored. For example, NPs that degrade into acidic products [16,17] and activate with light to generate acid [15] are reported. With regards to the former approach, poly(lactic-co-glycolic acid) (PLGA) NPs are able to restore lysosomal pH in retinal pigment epithelium (RPE) cells [16] and ATP13A2 cells, [17] thereby leading to restoration of autophagic flux and cellular function. The current study is the first to report the use of NPs to alter lysosomal dysfunction in pancreatic beta cells under lipotoxicity. Additionally, the biology of the pancreatic beta cell type is different than RPE and ATP13A2 cells, and hence the response(s) to NP treatment will afford new insights. We hypothesize that the PLGA NPs will acidify lysosomes in a NP concentration dependent manner and result in lowering of lysosomal pH, rescuing of autophagic flux, and restoration of glucose stimulated insulin secretion in β-cells, specifically rat pancreatic insulin secreting (INS1) β-cells. Results and Discussion Synthesis and Characterization of PLGA NPsMonodisperse PLGA NPs were formed using a previously reported sonication method using PLGA (Resomer 503 H) with sodium dodecyl sulfate (SDS) as the surfactant. [16] The PLGA NPs exhibit average diameters of 100 nm and polydispersity of 0.13 by dynamic light scattering (DLS) and possess a zeta Chronic exposure to high levels of fatty acids (lipotoxicity) in pancreatic beta cells (β-cells) decreases lysosomal acidity and inhibits autophagic flux. Today, there are a lack of approaches to modify lysosomal acidity to determine whether impaired lysosomal acidification is causally inhibiting autophagic flux and cellular functions. Biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) with diameters of ≈100 nm localize to lysosomes and serve as an ideal method to deliver lactic and glycolic acid to lysosomes upon NP polymer degradation. In this study, the ability of PLGA NPs to lower lysosomal pH and restore autophagic flux is investigated in pancreatic insulin secreting (INS1) β-cells. PLGA NPs display a concentration d...

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