Production of Bio-active Nanoparticle from Human Corneal Extracellular Matrix

Abstract

Purpose : Extracellular matrix (ECM) is composed of structural proteins and glycans as well as bio-active molecules such as growth factors, peptides and nucleic acids. Recent studies have shown the regenerative potential of processed decellularized ECMs derived from different organs. Here, we propose a novel protocol to produce nanoparticles with preserved bioactivity from human corneas for future corneal regenerative and tissue engineering applications.

Methods : Human cadaver corneas were kindly provided by Eversight Eye Bank. After washing, the corneas were cut into small pieces and decellularized by several cycles of freeze-thawing and nuclease treatment. The decellularized cornea pieces were then lyophilized and cryo-milled into microparticles (<230 µm particle size). The ECM microparticles were turned into nanoparticles by stirring in 5% acetic acid (5 mg/ml) followed by probe sonication. Various durations of acid treatment and sonication were applied to optimize the protocol. The particle sizes were measured using Dynamic Light Scattering. The proliferation of Human Corneal Epithelial Cells (HCECs) treated with 0.5 mg/ml human cornea ECM (HC-ECM) nanoparticles compared to control was assessed using a Cell Counting Kit to evaluate the bioactivity of produced samples.

Results : The average size of HC-ECM nanoparticles progressively decreased with increased time of acetic acid treatment (1645.3±373.5 nm, 843.5±301.4 nm and 390.9±331.4 nm after 24, 96 and 168 h, respectively). However, the mean polydispersity index (PDI) increased which indicates the reduction in uniformity of produced nanoparticles (0.85±0.16, 0.92±0.12 and 1±0.5 after 24, 96 and 168 h, respectively). Probe sonication not only decreased the size of particles but also improved the PDI in time (size, 493.7± 15.8nm, 485.8± 86.8nm, 314.3± 44.6nm and PDI, 0.58± 0.04, 0.4± 0.06, 0.48± 0.01 after 10, 20 and 30 minutes, respectively). We found that 24 hours stirring in 5% acetic acid followed by probe sonication for 20 minutes is the optimized protocol to produce ECM nanoparticles with highest uniformity. The proliferation of HCECs was significantly improved after treatment with HC-ECM nanoparticles compared to controls (P<0.001).

Conclusions : The proposed protocol for producing bioactive HC-ECM nanoparticles provides the potential opportunity of recycling unsuitable human corneas, for transplantation, into the therapeutic applications.

This is a 2020 ARVO Annual Meeting abstract.