Characterization of Oxygen Nanobubbles and In Vitro Evaluation of Retinal Cells in Hypoxia

Abstract

Purpose: Vein or artery occlusion causes a hypoxic environment by preventing oxygen delivery and diffusion to tissues. Diseases such as retinal vein occlusion, central retinal artery occlusion, or diabetic retinopathy create a stroke-type condition that leads to functional blindness in the effected eye. We aim to develop an oxygen delivery system consisting of oxygen nanobubbles (ONBs) that can mitigate retinal ischemia during a severe hypoxic event such as central retinal artery occlusion.

Methods: ONBs were synthesized to encapsulate oxygen saturated molecular medical grade water. Stability, oxygen release, biocompatibility, reactive oxygen species, superoxide, MTT, and terminal uridine nick-end labeling assays were performed. Cell viability was evaluated, and safety experiments were conducted in rabbits.

Results: The ONBs were approximately 220 nm in diameter, with a zeta potential of -58.8 mV. Oxygen release studies indicated that 74.06 µg of O2 is released from the ONBs after 12 hours at 37°C. Cell studies indicated that ONBs are safe and cells are viable. There was no significant increase in reactive oxygen species, superoxide, or double-stranded DNA damage after ONB treatment. ONBs preserve mitochondrial function and viability. Histological sections from rabbit eyes indicated that ONBs were not toxic.

Conclusions: The ONBs proposed have excellent oxygen holding and release properties to mitigate ischemic conditions in the retina. They are sterile, stable, and nontoxic.

Translation relevance: ONB technology was evaluated for its physical properties, oxygen release, sterility, stability, and safety. Our results indicate that ONBs could be a viable treatment approach to mitigate hypoxia during ischemic conditions in the eye upon timely administration.

Figure 1.

ONB size. Size distribution of ONBs measured by dynamic light scattering (A). TEM image of a typical dextran ONB (B).

Figure 2.

Oxygen release profile at varying temperatures. Oxygen concentration monitoring in hypoxia chamber at 37°C and room temperature of mixtures of ONBs.

Figure 3.

Oxygen release profiles at varying pHs. (A) Change in oxygen concentration upon release in isolated hypoxic system. The black line represents the average oxygen concentration, and the grey area depicts the standard deviation from 3 replications. (B) Oxygen concentration change with respect to pH. The pH of the sample in water is 7.0.

Figure 4.

Sterility and ROS evaluation in retinal cells. (A) Optical density of NBs in the soybean-casein digest media with bacteria over time (n = 5). (B) ROS generation after exposure to R28 and Muller cells with different concentration of ONBs (n = 8). (C) Superoxide levels in retinal cells after treatment (n = 8). NT, no treatment; Negative control, N-acetyl-L-cysteine; Positive control, pyocyanin. All data shown as mean ± standard deviation. ***P < 0.0001; *P < 0.05.

Figure 5.

Uptake of ONBs by retinal cells. (A) Quantitative representation of fluorescent acridine orange labeled ONBs (n = 4). NT, No treatment. All data are represented as the mean ± standard deviation. (B) Darkfield images of Muller cells and R28 cells. Cellular uptake of fluorescent NBs (green). Scale bar, 10 µm. Original magnification ×40.

Figure 6.

Viability of retinal cells post NB treatment. (A) MTT Viability assay of R28 and Muller cells after treatment with oxygen loaded NBs for 24 hours in normoxia. NT, No treatment. Data are shown as the mean ± standard deviation (n = 8). Representative images of terminal uridine nick-end labeling assay of Muller cells (B) or R28 cells (C) after 24 hours of exposure of O₂-loaded NBs at 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, Shell at 0.5 mg/mL (no O₂), no treatment, and positive control of TACS-nuclease treated cells. Scale bar, 5 µm.

Figure 7.

Viability of retinal cells in hypoxia with ONBs. Muller and R28 retina cell viability after 6 hours in hypoxia (5% O₂) with varying concentrations of NBs or no treatment. Shell (no O₂) groups were treated with a concentration of 0.5 mg/mL. Data are shown as mean ± standard deviation. NT, no treatment. † Significant difference to NT Normoxia. ‡ Significant difference to NT hypoxia (n = 8). ****P < 0.00001; ***P < 0.0001; **P < 0.001.

Figure 8.

Maximal mitochondria respiration after hypoxia and ONB treatment. After exposing R28 and Muller cells to 6 hours of hypoxia with three concentrations of oxygen-loaded dextran NBs, a dose-dependent protection of mitochondria maximal respiration was observed. Cells were seeded at 5000 cells/well and treated with an FCCP concentration of 1.5 µM. NT, no treatment. Data are shown as the mean ± standard deviation, with n = 8. ****P < 0.00001; ***P < 0.0001.

Figure 9.

Safety of ONB in vivo. (A) Change in the IOP of rabbit eyes after injection of saline or ONBs. Data shown as mean ± standard deviation with n = 6. (B) Representative images of rabbit retinas 7 days after injection. Rabbits were intravitreally injected with either saline (top: 101L, 102L, 103L) or ONBs (bottom: 104L, 105L, 106L). Scale bar, 20 µm. Original magnification ×40.