Toxicity of a stable phospholipid mixture on human retinal pigment epithelium cells

Abstract

Purpose : Microbubble-enhanced ultrasound has been proposed as a method to achieve targeted ocular drug delivery. We designed an in vitro experiment to identify the optimal concentration of a stable phospholipid mixture that biochemically recapitulates a monodisperse microbubble suspension and is nontoxic to human retinal epithelium cells (ARPE-19). We hypothesize that phospholipid mixture exposure reduces ARPE-19 cell viability in a dose-dependent fashion.

Methods : ARPE-19 cells were plated in 96-well plates and treated with 880 μL of solution containing 0.0125, 0.025, 0.05, 0.1, or 0.5 mg/mL of phospholipid mixture. Phospholipid was dissolved in chloroform at a 80:10:10 molar ratio of DPPC:DPPA:DPPE-PEG5000 (Avanti Polar Lipids) and dried by nitrogen stream and vacuum evaporation before resuspension in PBS. Control wells received media only. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay was performed at 24 and 48 hours post-treatment to quantify intracellular metabolism as a proxy for cell viability. All groups were run as 4 technical replicates and two-tailed Mann-Whitney U tests were used for statistical analysis.

Results : At 24 hours post-treatment, ARPE-19 cells treated with phospholipid mixture at concentrations at or above 0.05 mg/mL exhibited statistically significant (p<0.05) decreases in viability while cells treated with phospholipid mixture at concentrations below 0.05 mg/mL exhibited non-significant decreases in viability (Figure 1). This trend appeared to be dose-dependent. Only phospholipid mixture concentrations between 0.0125 mg/mL and 0.05 mg/mL were assessed in the 48-hour experiment; there were no statistically significant changes in cell viability between control and treatment groups in the 48-hour experiment (Figure 2).

Conclusions : This study demonstrates that short-term phospholipid exposure in ARPE-19 cells is cytotoxic in a dose-dependent fashion. This observation suggests that microbubble formulations should be fabricated below a certain phospholipid concentration to avoid cytotoxicity in target tissues. Future experiments evaluating phospholipid and microbubble toxicity in different ocular cell types are underway to more clearly characterize their physiological effects and explore the viability of microbubbles as an ocular drug delivery modality.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Relative cell viability (MTT) at 24 hours post-treatment

 

Relative cell viability (MTT) at 48 hours post-treatment