Tracking the heat-triggered phase change of polydopamine-shelled, perfluorocarbon emulsion droplets into microbubbles using neutron scattering

https://doi.org/10.1016/j.jcis.2021.08.162Get rights and content

Abstract

Perfluorocarbon emulsion droplets are hybrid colloidal materials with vast applications, ranging from imaging to drug delivery, due to their controllable phase transition into microbubbles via heat application or acoustic droplet vapourisation. The current work highlights the application of small- and ultra-small-angle neutron scattering (SANS and USANS), in combination with contrast variation techniques, in observing the in situ phase transition of polydopamine-shelled, perfluorocarbon (PDA/PFC) emulsion droplets with controlled polydispersity into microbubbles upon heating. We correlate these measurements with optical and transmission electron microscopy imaging, dynamic light scattering, and thermogravimetric analysis to characterise these emulsions, and observe their phase transition into microbubbles. Results show that the phase transition of PDA/PFC droplets with perfluorohexane (PFH), perfluoropentane (PFP), and PFH–PFP mixtures occur at temperatures that are around 30–40 °C higher than the boiling points of pure liquid PFCs, and this is influenced by the specific PFC compositions (perfluorohexane, perfluoropentane, and mixtures of these PFCs). Analysis and model fitting of neutron scattering data allowed us to monitor droplet size distributions at different temperatures, giving valuable insights into the transformation of these polydisperse, emulsion droplet systems.

Introduction

Phase-shifting or phase-change emulsion droplets contain low-boiling point perfluorocarbon (PFC) cores, stabilised by various types of shell materials, such as phospholipids, proteins, surfactants, and polymers [1], [2]. Common PFCs utilised in phase-change emulsion systems include perfluorohexane (C6F14, PFH), perfluoropentane (C5F12, PFP), decafluorobutane (C4F10, DFB), perfluorooctlylbromide (C8F17Br, PFOB), and octafluoropropane (C3F8, OFP) [2], [3], [4]. Phase-change emulsions display higher stability in physiological environment and the smaller size of these droplets make them superior carriers for intravascular delivery of therapeutics, when compared to microbubbles [5], [6]. Due to their size, phase-change emulsion droplets have more potential to leave the vascular compartment and be localised in the target site of action, including tumours, before transitioning into microbubbles, via acoustic droplet vapourisation (ADV) or via heat-induced vapourisation (hyperthermia) [4], [7], [8]. This phase change and concomitant formation of microbubbles have vast biomedical potential, ranging from contrast-enhanced vascular imaging to gas and drug delivery applications [2], [8], [9].

Potential stabilisers and coatings, such as polymers, proteins, and surfactants, can confer valuable physicochemical and biological properties to these droplets and, thereby, increase the versatility of these materials [10], [11], [12]. Polydopamine (PDA) has been demonstrated as a suitable, biocompatible stabiliser or shell material for phase-change emulsion droplets [1], [11], [13]. PDA forms via the oxidative polymerisation of dopamine at slightly basic pH, and adsorbs onto virtually any type and shape of surface and interface [14], [15], [16]. Reports in literature highlight PDA as a suitable shell for PFC droplets with many opportunities for further functionalisation, due to the multiple hydroxyl and amino groups present in the PDA structure. It is also an ideal coating material for intravenous carriers, since PDA possesses biocompatibility and antifouling properties [17], [18]. It can inhibit coated materials from interacting with biological matrix components, especially proteins that can alter their biological fate, and in consequence, their designed role or activity [19].

The study of these emulsion droplets and their heat-triggered phase change is of significant technical value, but has been proven to be challenging with conventional imaging and scattering techniques. Optical and electron microscopy (EM) imaging are both powerful tools in revealing PFC droplet and bubble structures. However, because of the inherent limit of resolution of optical imaging techniques [20] and the size and refractive index differences between the emulsion droplets and microbubbles, the use of optical microscopy is limited only to visualising large droplets and/or bubbles arising from micro- and submicron-sized PFC droplets [21], [22], [23], [24]. While previous studies have reported the application of various transmission electron microscopy imaging techniques in visualising emulsion droplets [1], [10], [22], [25] and microbubbles [26], [27], there has been no report on monitoring in situ the transformation of emulsion droplets into microbubbles. Furthermore, the harsh operational conditions (high vacuum and possibility of electron beam damage [28]) and limitations in applying various stimuli to induce phase change, restrict EM imaging from being a useful technique in observing this phase transition in real time. In using dynamic light scattering (DLS), an obstacle in exploring these colloidal systems is the considerable difference in densities, refractive indexes, and sizes of the droplets and bubbles, making it almost impossible to analyse these two materials simultaneously in aqueous environments.

Previous studies have demonstrated the use of small-angle and ultra-small-angle neutron scattering (SANS and USANS, respectively) in observing emulsion droplet structures [29], [30] and changes in different emulsion systems [31], [32]. These techniques use the elastic scattering of neutrons at small scattering angles to probe the structures of various materials on the lengthscale 1–500 nm for SANS and 100 nm–10 µm for USANS. Sample contrast in neutron scattering arises from the differences in the scattering length densities (SLD) of sample components, eliminating problems that are encountered in optical imaging and light scattering techniques. SANS and USANS also do not use harsh sample environment conditions, such as high or ultra-high vacuum that is needed in EM imaging, and are compatible with phase transition triggers, such as temperature [33] and ultrasound [34], and other stimuli, including shear [35] and magnetism [36], [37]. Moreover, because of the dependence of these techniques on the SLDs of the samples, by varying the proton-to-deuterium ratios of the dispersing medium, there is an opportunity for contrast variation: making one component or phase transparent to neutrons to highlight the scattering from another [38]. Despite these advantages of SANS and USANS, only a limited number of reports in the literature describe the use of these techniques in studying microbubbles and the phase transition of phase-change emulsion droplets into microbubbles.

In this study, we utilise a variety of techniques, including conventional optical microscopy and EM imaging, dynamic light and neutron scattering techniques (Fig. 1), and thermogravimetric analysis to study in situ the temperature-dependent phase change of polydopamine-shelled, perfluorocarbon (PDA/PFC) emulsion droplets with different PFH-to-PFP ratios into microbubbles. This work highlights the advantages of applying SANS and USANS, with contrast variation techniques to separate the scattering of droplets and microbubbles, and particularly the application of a tumbling system to address issues with gravitational sedimentation/buoyancy, while observing the overall properties of both the droplets and bubbles before, during, and after phase transition.

Section snippets

Preliminary characterisation of PDA/PFC droplets

PDA/PFC droplets with different mass ratios of PFC cores (PFH, PFP, and mixtures of both) were fabricated via ultrasonic emulsification, followed by oxidative polymerisation of the PDA shell [1]. In this process, Pluronic F-127 at low concentrations acted as the emulsion stabiliser while allowing PDA to adsorb onto the droplets’ interface to form a rigid polymer shell. The PDA shell is the main stabiliser of the emulsion droplets since: (1) a fully formed PDA shell is expected to confine and

Conclusion

Contrast variation small- and ultra-small-angle scattering techniques, combined with temperature control and a tumbling system, were used to study the changes in droplet size and interfacial structures of the PDA/PFC droplets. These studies were supported by a combination of transmission electron and optical microscopy imaging, dynamic light scattering, and thermogravimetric analysis. Based on the results of the experiments, we found that PDA/PFC droplets undergo an abrupt phase change into

Materials and reagents

Dopamine hydrochloride (C8H11NO7 HCl, Sigma-Aldrich), copper sulfate pentahydrate (CuSO4·5H2O, Sigma-Aldrich), Pluronic F-127 (Sigma-Aldrich), tris(hydroxymethyl)-aminomethane hydrochloride (TRIS HCl, ultrapure, VWR Life Science), perfluoropentane (C5F10, Synquest Laboratories) and perfluorohexane (C6F14, FluoroChem) were used as received.

Preparation of PDA/PFC emulsion droplets

The method for the preparation of the PDA/PFH droplets were reported in previous works [1]. A mixture, containing 14.00 mL Tris buffer (10 mol L−1, pH 8.5),

CRediT authorship contribution statement

Mark Louis P. Vidallon: Conceptualization, Methodology, Investigation, Visualization, Writing - original draft. Luke W. Giles: Investigation, Visualization, Writing - review & editing. Matthew J. Pottage: Investigation, Writing - review & editing. Calum S.G. Butler: Investigation, Writing - review & editing. Simon A. Crawford: Investigation. Alexis I. Bishop: Methodology, Resources, Supervision, Writing - review & editing. Rico F. Tabor: Conceptualization, Methodology, Resources, Supervision,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors acknowledge the support of the Australian Nuclear Science and Technology Organisation (ANSTO), in providing the Bilby SANS and Kookaburra USANS instruments and facilities used in this work (P8365). The authors also acknowledge the use of equipment at Monash Centre for Electron Microscopy (MCEM), Ramaciotti Centre for Cryo-Electron Microscopy, and Monash Analytical Platform. The authors acknowledge the technical support from Mr. Scott Blundell during the COVID19 pandemic. The authors

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