Abstract
Herein we showcase the potential of ultrasound-responsive nanobubbles in enhancing macromolecular permeation through layers of the retina, ultimately leading to significant and direct intracellular delivery; this being effectively demonstrated across three relevant and distinct retinal cell lines. Stably engineered nanobubbles of a highly homogenous and echogenic nature were fully characterised using dynamic light scattering, B-scan ultrasound and transmission electron microscopy (TEM). The nanobubbles appeared as spherical liposome-like structures under TEM, accompanied by an opaque luminal core and darkened corona around their periphery, with both features indicative of efficient gas entrapment and adsorption, respectively. A nanobubble +/- ultrasound sweeping study was conducted next, which determined the maximum tolerated dose for each cell line. Detection of underlying cellular stress was verified using the biomarker heat shock protein 70, measured before and after treatment with optimised ultrasound. Next, with safety to nanobubbles and optimised ultrasound demonstrated, each human or mouse-derived cell population was incubated with biotinylated rabbit-IgG in the presence and absence of ultrasound +/- nanobubbles. Intracellular delivery of antibody in each cell type was then quantified using Cy3-streptavidin. Nanobubbles and optimised ultrasound were found to be negligibly toxic across all cell lines tested. Macromolecular internalisation was achieved to significant, yet varying degrees in all three cell lines. The results of this study pave the way towards better understanding mechanisms underlying cellular responsiveness to ultrasound-triggered drug delivery in future ex vivo and in vivo models of the posterior eye.
Highlights
Pathologies of the retina continue to pose an ominous burden on healthcare systems globally with conditions such as age-related macular degeneration (AMD), glaucoma and diabetic retinopathies (DR) listed among the top 10 priority eye diseases by the World Health Organization [1]
As ultrasound administration will likely subject a broad region of the tissue to similar mechanical and thermal stressors, we conducted evaluations on three different cell lines which represented unique populations found within the retina i.e. epithelial (ARPE-19), glial (MIO-M1), and neuronal (661W) cell types
This study evaluated the impact of a novel nanobubble-ultrasound strategy on three distinct retinal cell lines
Summary
Pathologies of the retina continue to pose an ominous burden on healthcare systems globally with conditions such as age-related macular degeneration (AMD), glaucoma and diabetic retinopathies (DR) listed among the top 10 priority eye diseases by the World Health Organization [1]. While various promising therapeutic agents have been developed in recent years, an overwhelming bottleneck to their utility remains an inability to preferentially deliver them into target tissue/cells of the posterior eye with any level of precision or accuracy [2] This is in part due to the remote and highly inaccessible location of the affected retinal tissue, which is multi-layered and comprising many associated protective barriers. Micro- or nano-sized contrast agents entrapping gas within a surfactant-based shell oscillate through cycles of expansion and contraction, this in response to ultrasound In this context ultrasound can be used to rupture/implode the bubbles by a phenomenon known as inertial cavitation, which can generate microjets resulting in the propulsion of co-delivered therapeutics deep into surrounding cells/tissue [3]. Given the likely differences in intercellular sensitivity to the effects of ultrasound-assisted bubble cavitation, broader evaluation of co-localised cell types is expected to provide a more holistic understanding of the impact that ultrasound-assisted administration of our nanobubbles will have on representative cells of the retina [4, 5, 8,9,10, 19]
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