Probing phospholipid microbubbles by atomic force microscopy to quantify bubble mechanics and nanostructural shell properties

Abstract

Highlights  Using tapping mode imaging, the thickness of microbubble shells can be quantified  Quantifying shell thickness has significant impact on Young's Modulus values  Atomic force microscopy can be used to quantify thickness of the PEG brush layer ABSTRACT: Microbubbles (MBs), which are used as ultrasonic contrast agents, have distinct acoustic signatures which enable them to significantly enhance visualisation of the vasculature. Research is progressing to develop MBs which act as drug/gene delivery vehicles for site-specific therapeutics. In order to manufacture effective theranostic vehicles, it is imperative to understand the mechanical and nanostructural properties of these agents; this will enrich the understanding of how the structural, biophysical and chemical properties of these bubbles impact their functionality. We produced microfluidic phospholipid-based MBs due to their favourable properties, such as biocompatibility and echogenicity, as well as the ability to modify the shell for targeting applications. We have drawn upon atomic force microscopy to conduct force-spectroscopy and tapping-mode imaging investigations. We have, for the first time to our knowledge, been able to accurately quantify the thickness and lipid configuration of phospholipid-shelled MBs-showing a trilayer as opposed to the conventional monolayer structure. Furthermore, we have measured MB stiffness and employed different mechanical theories to quantify the Young s Modulus. We show that the Reissner theory is inappropriate for mechanical characterisation of phospholipid MBs, however, the Hertz model does offer biologically relevant comparisons. Analysis using the Alexander-de Gennes polymer brush theory has allowed us to provide new information regarding how the thickness of the polyethylene glycol brushes, end-grafted to our phospholipid microbubbles, changes with diameter.

Freeze-Dried Therapeutic Microbubbles: Stability and Gas Exchange

“…A Horizon instrument (Horizon v3, Leeds, UK) was used for all MB production with a polymethylmethacrylate (PMMA) flow-focused MF chip designed in-house and fabricated by Epigem (Redcar, UK) to prepare MBs according to methodologies previously published. , Both the liquid flow rate and gas (perfluorobutane (C 4 F 10 ) from F2 chemicals (Preston, UK)) pressure were controlled by a p-pump, a pressure-based pumping system from Dolomite (Royston, UK).…”

Spherical oscillations of encapsulated microbubbles: Effect of shell compressibility and anisotropy

“…Physically, √ ε is the ratio of the typical time scale τ 0 = d e ρ s /E needed for an elastic wave to travel across the shell thickness d e over the time scale ω −1 0 of the motion of the boundary. In general, E is not smaller than 100 MPa for a polymeric material where d e ∼ 100 nm, but for lipid shells of thickness of order 5 nm which are made of the type of anisotropic material that we treat later on in this paper, orders of magnitude as low as 100 kPa were proposed for an effective isotropic Young modulus (Shafi et al, 2019).…”

“…The atomic force microscopy (AFM) is a direct approach used to estimate UCAs' properties. However, depending on the model used to extract elastic constants from the force-displacement curve of an AFM, very different values can be found (Abou-Saleh et al, 2013;Buchner Santos et al, 2012;Lytra et al, 2020;Shafi et al, 2019). This makes the validation of spherical oscillation models a tricky task so far.…”

“…While commercial UCAs are quite polydisperse in size, narrowing the size distribution of UCAs appears then as a way to match better the relatively narrow frequency bandwidth of ultrasonic devices with that of the UCAs, thus leading to better sensitivity of the whole detection process. Recent works go in that direction, that make use of shell material of various types, such as polymers (Liu et al, 2014;Song et al, 2018), phospholipids (Gong et al, 2014;Helfield et al, 2014;Lum et al, 2016;Parrales et al, 2014;Segers et al, 2016Segers et al, , 2020van Rooij et al, 2015) -sometimes forming more than 2 layers (Shafi et al, 2019), silica (Hu et al, 2011) or proteins (Wang et al, 2020). This calls for models of bubble oscillations that are able to describe a wide variety of shell materials.…”

Radial dynamics of an encapsulated microbubble with interface energy

“…Though EBs are widely used for ultrasound medical imaging, in particular, a recent study Errico et al (2015) have shown that these bubbles can not only be used as rough contrast agents, rather can also be used as traces to extract vascular geometry (microvascular imaging) and blood flow velocity over a large dynamic range in microns length scales. The encapsulation of such bubbles can be made up of polymers (Liu et al 2014;Song et al 2018), proteins (Wang et al 2020) or phospholipids (Santos et al 2012;Li et al 2013a;Abou-Saleh et al 2013;Gong et al 2014;Parrales et al 2014;van Rooij et al 2015;Segers et al 2016;Lum et al 2016;Helfield 2019;Shafi et al 2019;Segers et al 2020). Thus experimental and theoretical understanding of the response of EBs to the external acoustic field is of great importance for the future developments of these promising applications/techniques.…”