Elsevier

Ultrasonics Sonochemistry

Volume 66, September 2020, 105109
Ultrasonics Sonochemistry

Standing wave-assisted acoustic droplet vaporization for single and dual payload release in acoustically-responsive scaffolds

https://doi.org/10.1016/j.ultsonch.2020.105109Get rights and content

Highlights

  • ADV enables controlled payload release from acoustically-responsive scaffolds (ARSs).

  • An ultrasound standing wave field (SWF) enhanced payload release from ARSs.

  • SWF-assisted ADV enabled sequential release of two payloads from bi-layer ARSs.

  • Bubble morphology was dependent on the presence of standing waves.

  • The scaffold degradation rate decreased following ultrasound exposure.

Abstract

An ultrasound standing wave field (SWF) has been utilized in many biomedical applications. Here, we demonstrate how a SWF can enhance drug release using acoustic droplet vaporization (ADV) in an acoustically-responsive scaffold (ARS). ARSs are composite fibrin hydrogels containing payload-carrying, monodispersed perfluorocarbon (PFC) emulsions and have been used to stimulate regenerative processes such as angiogenesis. Elevated amplitudes in the SWF significantly enhanced payload release from ARSs containing dextran-loaded emulsions (nominal diameter: 6 μm) compared to the -SWF condition, both at sub- and suprathreshold excitation pressures. At 2.5 MHz and 4 MPa peak rarefactional pressure, the cumulative percentage of payload released from ARSs reached 84.1 ± 5.4% and 66.1 ± 4.4% under + SWF and -SWF conditions, respectively, on day 10. A strategy for generating a SWF for an in situ ARS is also presented. For dual-payload release studies, bi-layer ARSs containing a different payload within each layer were exposed to temporally staggered ADV at 3.25 MHz (day 0) and 8.6 MHz (day 4). Sequential payload release was demonstrated using dextran payloads as well as two growth factors relevant to angiogenesis: basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB (PDGF-BB). In addition, bubble growth and fibrin degradation were characterized in the ARSs under +SWF and -SWF conditions. These results highlight the utility of a SWF for modulating single and dual payload release from an ARS and can be used in future therapeutic studies.

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.

References (67)

  • F. De Marchis

    Platelet-derived growth factor inhibits basic fibroblast growth factor angiogenic properties in vitro and in vivo through its α receptor

    Blood

    (2002)
  • A. Moncion

    In vitro and in vivo assessment of controlled release and degradation of acoustically responsive scaffolds

    Acta Biomater.

    (2016)
  • M. Aliabouzar

    Acoustic droplet vaporization in acoustically responsive scaffolds: effects of frequency of excitation, volume fraction and threshold determination method

    Ultrasound Med. Biol.

    (2019)
  • S.E. Sakiyama-Elbert

    Controlled-release kinetics and biologic activity of platelet-derived growth factor-BB for use in flexor tendon repair

    J. Hand Surg.

    (2008)
  • N.S. Patil et al.

    Macroporous poly (sucrose acrylate) hydrogel for controlled release of macromolecules

    Biomaterials

    (1996)
  • K. Hensel et al.

    Analysis of ultrasound fields in cell culture wells for in vitro ultrasound therapy experiments

    Ultrasound Med. Biol.

    (2011)
  • J. Weisel

    Stressed fibrin lysis

    J. Thromb. Haemost.

    (2011)
  • I. Varjú

    Hindered dissolution of fibrin formed under mechanical stress

    J. Thromb. Haemost.

    (2011)
  • O.V. Kim

    Structural basis for the nonlinear mechanics of fibrin networks under compression

    Biomaterials

    (2014)
  • W. Liu

    The mechanical properties of single fibrin fibers

    J. Thromb. Haemost.

    (2010)
  • N.E. Hudson

    Stiffening of individual fibrin fibers equitably distributes strain and strengthens networks

    Biophys. J.

    (2010)
  • C. Cha et al.

    Tuning the dependency between stiffness and permeability of a cell encapsulating hydrogel with hydrophilic pendant chains

    Acta Biomater.

    (2011)
  • R.G. Holt et al.

    Measurements of bubble-enhanced heating from focused, MHz-frequency ultrasound in a tissue-mimicking material

    Ultrasound Med. Biol.

    (2001)
  • Y. Du

    Convection-driven generation of long-range material gradients

    Biomaterials

    (2010)
  • M.T. Burgess et al.

    Control of acoustic cavitation for efficient sonoporation with phase-shift nanoemulsions

    Ultrasound Med. Biol.

    (2019)
  • N. Reznik et al.

    Investigation of vaporized submicron perfluorocarbon droplets as an ultrasound contrast agent

    Ultrasound Med. Biol.

    (2011)
  • M.L. Fabiilli

    Delivery of water-soluble drugs using acoustically triggered perfluorocarbon double emulsions

    Pharm. Res.

    (2010)
  • B.A. Juliar

    In situ transfection by controlled release of lipoplexes using acoustic droplet vaporization

    Adv. Healthcare Mater.

    (2016)
  • X. Dong

    Controlled delivery of basic fibroblast growth factor (bFGF) using acoustic droplet vaporization stimulates endothelial network formation

    Acta Biomater.

    (2019)
  • K.-i. Kawabata, et al. 1F-3 Site-Specific Contrast Imaging with Locally Induced Microbubbles from Liquid Precursors. in...
  • C.R. Courtney

    Manipulation of microparticles using phase-controllable ultrasonic standing waves

    J. Acoust. Soc. Am.

    (2010)
  • A.A. Ucuzian

    Molecular mediators of angiogenesis

    J. Burn Care Res.

    (2010)
  • P. Carmeliet et al.

    Molecular mechanisms and clinical applications of angiogenesis

    Nature

    (2011)
  • Cited by (19)

    • Multi-time scale characterization of acoustic droplet vaporization and payload release of phase-shift emulsions using high-speed microscopy

      2022, Ultrasonics Sonochemistry
      Citation 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, Bioprinting
      Citation 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.

    View all citing articles on Scopus
    View full text