Stably engineered nanobubbles and ultrasound - An effective platform for enhanced macromolecular delivery to representative cells of the retina

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.

Cavitation-threshold Determination and Rheological-parameters Estimation of Albumin-stabilized Nanobubbles

“…This includes liposomes (lipid bilayer shell including an aqueous core with gas pockets) 39 – 41 , cavitation seeds acting as gas-pocket traps sur as nanocups 42 or porous nanoparticles 43 , NBs of lipids, polymers, or protein shells with a gaseous core 44 – 48 , and nanodroplets (lipid or polymer shell with the specificity of being phase-changing agents) 49 . There are numerous applications for which NBs provide interesting results, notably drug delivery to various targets: tumor 50 – 58 , nerve 59 , retina 60 , vascular tissues 61 , brain blood barrier 62 . They can also be used to perform gene transfection 63 – 68 .…”

Development of a lipoid microbubble formulation and in vitro evaluation assisted by ultrasound

“…900 µL of the supernatant was carefully collected (labelled as A in Figure 3 This effect is reported to be due to higher cellular uptake of the drug, caused by cavitation of the bubbles, leading to higher concentrations of intracellular doxycycline. Therefore, bacteria present inside host mammalian cells are exposed to a higher dose of doxycycline, correlating with previous reports of enhanced cellular uptake of different drugs using US-induce cavitation (144)(145)(146)(147).…”

“…The remote loadings were performed at 65 °C (using a Heidolph® incubator1000) and the liposomes (11mM) were made of DSPC: DSPEmPEG-2000 (94:6). o te lo a d in g tim e (m in u te ) % of loading efficiency in ciprofloxacin HCl remote loading technique, comparison between the 4 different remote loading times (i.e.5, 10, 20 and 30 min) performed at 65 ºC using Heidolph® incubator1000, the concentration of lipid solution was 11mM, liposomes were made of DSPC:DSPEmPEG-2000 at molar ratio of 94:6, the lipid film was hydrated by 135 mM ammonium sulphate solution, liposomes were prepared via extrusion and the PDI was ≤ 0.1 for all of the groups, the % of remote loading is determined via size exclusion chromatography assay, the liposomes were ruptured using Triton X-100, 0.5% v/v and the amount of liposomal ciprofloxacin was quantified by HPLC, the remote loading time with the highest % of lading efficiency is coloured in green, (n=3 independent experiments, data shown as mean ± SD) Supercharging of liposomesThe ciprofloxacin-filled liposomes were subjected to supercharging with PFP (US contrast agent), this being performed under pressure using an earlier standardized and established protocol(129). Briefly, the liposomal solution (2 mL) was transferred to an air evacuated 9 mL crimp sealed vial.…”

Cryo-EM Visualization of Lipid and Polymer-Stabilized Perfluorocarbon Gas Nanobubbles - A Step Towards Nanobubble Mediated Drug Delivery

“…We hypothesize that the dark cores observed in cryo-EM images of crosslinked and non-crosslinked nanobubbles are indicative of encapsulated octafluoropropane gas (molecular weight of 188 g/mol). We note that dark cores have recently been observed in cryo-EM images of a nanobubble formulation designed for enhanced macromolecular delivery to the retina 27 . A small percentage (<10%) of nanobubbles in our preparation are observed without the electron dense core and these are presumably empty or malformed nanobubbles (Fig.…”