Standing wave-assisted acoustic droplet vaporization for single and dual payload release in acoustically-responsive scaffolds
Introduction
Conventional hydrogel-based, drug delivery approaches are mainly governed by passive mechanisms like diffusion and degradation. Therefore, after implantation of a hydrogel scaffold, the release kinetics of deliverable therapeutic agents (e.g., growth factors) cannot be altered. Designing scaffold-based delivery systems that enable active modulation of release, via an externally-controlled mechanism, would be beneficial. Ultrasound (US)-triggered drug delivery can induce activation with temporal and spatial specificity, thereby providing non-invasive and on-demand control of release kinetics. One application where spatiotemporally-controlled release is critically important is tissue regeneration, since biological processes are regulated by the spatial profile and temporal expression of molecules like growth factors [1].
We have utilized acoustic droplet vaporization (ADV), a threshold-based phenomenon, to control release of therapeutic agents encapsulated in phase shift emulsions with a double emulsion structure of water-in-perfluorocarbon (PFC)-in-water (W1/PFC/W2) [2], [3]. The deliverable payload is contained within the innermost water phase (i.e., W1). US can disrupt the morphology of the double emulsion by phase-transitioning the PFC phase, thereby releasing the payload. Phase shift emulsions can be incorporated into implantable fibrin scaffolds to produce a composite hydrogel, termed an acoustically-responsive scaffold (ARS). ARSs have be used in conjunction with ADV in both in vitro [4] and in vivo [5] studies to control delivery of fluorescently-labeled dextran as well as basic fibroblast growth factor (bFGF). ADV-triggered delivery of bFGF stimulated endothelial network formation in vitro [6] and subcutaneous angiogenesis in vivo [7]. Given the necessity for multiple growth factors in tissue regeneration, ARSs have also been used for the sequential delivery of two surrogate payloads by encapsulating each payload in separate emulsions, each having a unique ADV threshold [8].
To optimize release of a therapeutic agent from an ARS, two criteria are important. First, the emulsion within the ARS should be thermally stable at body temperature, therefore only releasing its payload when exposed to suprathreshold US. Second, when exposed to suprathreshold US, the fraction of droplets that undergo ADV (i.e., the ADV efficiency) should be high. PFCs with higher bulk boiling points offer better thermal stability by eliminating any potential spontaneous vaporization as well as higher probability of repeated vaporization and re-condensation which are desired for extended diagnostic and therapeutic applications [9]. However, the ADV threshold correlates with the boiling point of the PFC in the emulsion. Thus, in order to achieve a high ADV efficiency using higher boiling point PFCs, greater acoustic input energies are required, which can limit their use in biomedical applications. Alternatively, creating an US standing wave field (SWF) can generate elevated amplitudes due to constructive superposition of a reflected wave at the interface of two media of differing acoustic impedances with an incident wave. A SWF has been used in biomedical applications including sonoporation-induced intracellular drug delivery [10], US-mediated gene delivery [11], cell banding [12], [13], and particle sorting [14]. Lo, Kripfgans [15] showed that creating a plane of pre-formed bubbles further enhanced ADV on the proximal side of the bubble wall.
In this work, we created a SWF to generate a suprathreshold pressure field at subthreshold excitation pressures. The acoustic pressure field was characterized under +SWF and –SWF (i.e., without establishment of standing waves in the US field) conditions. The effect of a SWF on the release profile of single-layer ARSs doped with dextran-containing monodispersed emulsions was investigated at varying peak rarefactional pressures. A strategy for generating a SWF for an ARS implanted in situ was also experimentally verified. Sequential release of two payloads was conducted by integrating the SWF with the frequency-dependence of the ADV threshold in a bi-layer ARS containing two dextran payloads. Similarly, we also demonstrated for the first time, sequential release of two growth factors relevant to angiogenesis, bFGF and platelet-derived growth factor BB (PDGF-BB), from bi-layer ARSs. bFGF is involved with the sprouting of new blood vessels while PDGF-BB stimulates neovessel stabilization via the recruitment of pericytes [16], [17]. bFGF and PDGF-BB are mutually antagonistic when delivered simultaneously [18], [19]. In addition, growth of the ADV-generated bubbles in ARSs was compared optically in the presence and absence of a SWF. Finally, the combined effect of ADV and subsequent bubble growth on the fibrin network degradation in ARSs was investigated.
Section snippets
Preparation and characterization of monodisperse double emulsions
Micron-sized, double emulsions with a W1/PFC/W2 structure were prepared using a microfluidic-based technique following a previously described method [7]. Perfluoropentane (C5, CAS# 678-26-2, bulk boiling point: 29 °C, Strem Chemicals, Newburyport, MA, USA), perfluorohexane (C6, CAS# 355-42-0, bulk boiling point: 56 °C, Strem Chemicals), or perfluorononane (C9, CAS# 375-96-2, bulk boiling point: 125 °C, Sigma-Aldrich, St. Louis, MO, USA) was used as the PFC phase. A fluorosurfactant copolymer,
Characterization of double emulsions
Sizing characteristics of the emulsions used in the ARSs are summarized in Table 2. There were no significant differences in mean diameter among the various PFC emulsions. C5-blank emulsions had a broader size distribution as well as coefficient of variation. Overall, the type of payload did not affect the size distribution of the double emulsions used in the ARSs.
ADV and IC thresholds
The ADV and IC thresholds of ARSs were determined through passive detection of the scattered response and the broadband acoustic
Conclusions
In this work, we first studied the impact of a SWF on payload release from single-payload containing ARSs using ADV. At 2.5 MHz (Pr = 4 MPa), elevated amplitudes due to the constructive superposition in the SWF increased payload release from C6 double emulsions by 21% on day 10 compared to the ARSs in -SWF condition. Fibrin degradation was shown to correlate inversely with the excitation amplitude, being highest for the sham ARSs and lowest for ARSs at 8 MPa in the SWF, likely due to hindered
CRediT authorship contribution statement
Mitra Aliabouzar: Conceptualization, Investigation, Formal analysis, Writing - original draft, Visualization. Aniket Jivani: Formal analysis, Investigation. Xiaofang Lu: Resources. Oliver D. Kripfgans: Conceptualization, Software. J. Brian Fowlkes: Conceptualization. Mario L. Fabiilli: Conceptualization, Resources, Supervision, Funding acquisition, Writing - review & editing.
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
This work was supported by NIH Grant R01HL139656 (M.L.F.). Special thanks to Dr. Allen Brooks (Department of Radiology) for assisting with the synthesis of the fluorosurfactant as well as the Fabrication Studio at the Duderstadt Center and Dr. William Weadock (Department of Radiology) for helping with 3D printing of materials related to the US exposure setup.
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2022, Ultrasonics SonochemistryCitation Excerpt :These higher molecular weight PFCs are more thermally stable, thus eliminating the potential for spontaneous bubble formation that can occur in implanted ARSs over the course of days or weeks [10]. Although we have utilized higher bulk boiling point PFCs in our prior in vitro and in vivo studies to control release of single payloads [7], sequential release of two payloads [11,10], as well as micro-patterned reservoirs of multiple payloads [12] in ARSs, little is known about their vaporization dynamics and release kinetics. Insight into the physics of vaporization will help formulation and selection of PFC emulsions for specific applications.
Micropatterning of acoustic droplet vaporization in acoustically-responsive scaffolds using extrusion-based bioprinting
2022, BioprintingCitation Excerpt :Sizing characteristics of the prepared PSEs are summarized in Table 1. The type of labeled dextrans in the W1 phase did not affect the size distribution of PSEs, similar to our previous publication [8]. Acoustically-responsive bioinks were prepared by first adding sodium alginate powder (alginic acid sodium salt, CAS# 9005-38-3, Sigma-Aldrich) to deionized (DI) water at 2–10% (w/v) and dissolving it overnight.