Acoustically-responsive Scaffolds Research Articles

Acoustically responsive scaffolds: Unraveling release kinetics and mechanisms for sustained, steady drug delivery

Hydrogels can serve as local drug delivery depots that protect the biological activity of labile therapeutics. However, drug release from conventional hydrogels is typically rapid, which is not ideal for many therapeutic agents. We developed a composite hydrogel that enables sustained drug release in response to ultrasound. The composite, termed an acoustically responsive scaffold (ARS), consists of a fibrin hydrogel and a phase-shift emulsion. Upon exposure to ultrasound, the emulsion is vaporized into bubbles, which leads to release of drugs contained within the emulsion. Previously, ARSs have been used in regenerative applications to stimulate blood vessel growth. Here, we characterize the release kinetics and mechanisms of ARSs. Release exhibits a triphasic pattern compromising a slow phase prior to ultrasound exposure; a transient, fast phase immediately after ultrasound exposure that follows a sigmoidal profile; and a sustained, steady phase. In each phase, we demonstrate how derived kinetics parameters are impacted by the ARS composition (e.g., fibrin and emulsion concentrations) and ultrasound properties (e.g., acoustic pressure, pulse duration). Using confocal microscopy, protein assays, and B-mode ultrasound imaging, we demonstrate that drug release from an ARS is independent of fibrin degradation and dependent on bubble growth. These results are critical in optimizing ARSs for delivery of therapeutic agents.

Acoustic droplet vaporization for on-demand modulation of microporosity in smart hydrogels.

Microporosity in hydrogels is critical for directing tissue formation and function. We have developed a fibrin-based smart hydrogel, termed an acoustically responsive scaffold (ARS), which responds to focused ultrasound in a spatiotemporally controlled, user-defined manner. ARSs are highly flexible platforms due to the inclusion of phase-shift droplets and their tunable response to ultrasound through a mechanism termed acoustic droplet vaporization (ADV). Here, we demonstrated that ADV enabled consistent generation of micropores in ARSs, throughout the entire thickness (∼5.5 mm), utilizing perfluorooctane phase-shift droplets. Size characteristics of the generated micropores were quantified in response to critical parameters including acoustic properties, droplet size, and shear elastic modulus of fibrin using confocal microscopy. The findings showed that the length of the generated micropores correlated directly with excitation frequency, peak rarefactional pressure, pulse duration, droplet size, and indirectly with the shear elastic modulus of the fibrin matrix. The ADV-generated micropores in ARSs were further compared with cavitation-mediated micropores in fibrin gels without droplets. Additionally, the Keller-Miksis equation was used to predict an upper bound for micropore formation in ARSs at varying driving frequencies and droplet sizes. Finally, our in vivo studies showed that host cell migration following ADV-induced micropore formation was frequency-dependent, with up to 2.6 times higher cell migration at lower frequencies. Overall, these findings demonstrate a new potential application of ADV in hydrogels. STATEMENT OF SIGNIFICANCE: Interconnected micropores within a hydrogel can facilitate many cell-mediated processes. Most techniques for generating micropores are typically not biocompatible or do not enable controlled, in situ micropore formation. We used an ultrasound-based technique, termed acoustic droplet vaporization, to generate microporosity in smart hydrogels termed acoustically responsive scaffolds (ARSs). ARSs contain a fibrin matrix doped with a phase-shift droplet. We demonstrate that unique acoustic properties of phase-shift droplets can be tailored to yield spatiotemporally controlled, on-demand micropore formation. Additionally, the size characteristics of the ultrasound-generated micropores can be modulated by tuning ultrasound parameters, droplet properties, and bulk elastic properties of fibrin. Finally, we demonstrate significant, frequency-dependent host cell migration in subcutaneously implanted ARSs in mice following ultrasound-induced micropore formation in situ.

Ultrasound-Induced Mechanical Compaction in Acoustically Responsive Scaffolds Promotes Spatiotemporally Modulated Signaling in Triple Negative Breast Cancer.

Cancer cells continually sense and respond to mechanical cues from the extracellular matrix (ECM). Interaction with the ECM can alter intracellular signaling cascades, leading to changes in processes that promote cancer cell growth, migration, and survival. The present study used a recently developed composite hydrogel composed of a fibrin matrix and phase-shift emulsion, termed an acoustically responsive scaffold (ARS), to investigate effects of local mechanical properties on breast cancer cell signaling. Treatment of ARSs with focused ultrasound drives acoustic droplet vaporization (ADV) in a spatiotemporally controlled manner, inducing local compaction and stiffening of the fibrin matrix adjacent to the matrix-bubble interface. Combining ARSs and live single cell imaging of triple-negative breast cancer cells, it is discovered that both basal and growth-factor stimulated activities of protein kinase B (also known as Akt) and extracellular signal-regulated kinase (ERK), two major kinases driving cancer progression, negatively correlate with increasing distance from the ADV-induced bubble both in vitro and in a mouse model. Together, these data demonstrate that local changes in ECM compaction regulate Akt and ERK signaling in breast cancer and support further applications of the novel ARS technology to analyze spatial and temporal effects of ECM mechanics on cell signaling and cancer biology.

Micropatterning of acoustic droplet vaporization in acoustically-responsive scaffolds using extrusion-based bioprinting

Acoustically-responsive scaffolds (ARSs) are composite hydrogels that respond to ultrasound in an on-demand, spatiotemporally-controlled manner due to the presence of a phase-shift emulsion. When exposed to ultrasound, a gas bubble is formed within each emulsion droplet via a mechanism termed acoustic droplet vaporization (ADV). In previous in vitro and in vivo studies, we demonstrated that ADV can control regenerative processes by releasing growth factors and/or modulating micromechanics in ARSs. Precise, spatial patterning of emulsion within an ARS could be beneficial for ADV-induced modulation of biochemical and biophysical cues. However, precise patterning is limited using conventional bulk polymerization techniques. Here, we developed an extrusion-based method for bioprinting ARSs with micropatterned structures. Emulsions were loaded within bioink formulations containing fibrin, hyaluronic acid and/or alginate. Experimental as well as theoretical studies elucidated the interrelations between printing parameters, needle geometry, rheological properties of the bioink, and the process-induced mechanical stresses during bioprinting. The shear thinning properties of the bioinks enabled use of lower extrusion pressures resulting in decreased shear stresses and shorter residence times, thereby facilitating high viability for cell-loaded bioinks. Bioprinting yielded greater alignment of fibrin fibers in ARSs compared to conventionally polymerized ARSs. Bioprinted ARSs also enabled generation of ADV at high spatial resolutions, which were otherwise not achievable in conventional ARSs, and acoustically-driven collapse of ADV-induced bubbles. Overall, bioprinting could aid in optimizing ARSs for therapeutic applications.

Spatiotemporal control of myofibroblast activation in acoustically-responsive scaffolds via ultrasound-induced matrix stiffening

Hydrogels are often used to study the impact of biomechanical and topographical cues on cell behavior. Conventional hydrogels are designed a priori, with characteristics that cannot be dynamically changed in an externally controlled, user-defined manner. We developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), that enables non-invasive, spatiotemporally controlled modulation of mechanical and morphological properties using focused ultrasound. An ARS consists of a phase-shift emulsion distributed in a fibrin matrix. Ultrasound non-thermally vaporizes the emulsion into bubbles, which induces localized, radial compaction and stiffening of the fibrin matrix. In this in vitro study, we investigate how this mechanism can control the differentiation of fibroblasts into myofibroblasts, a transition correlated with substrate stiffness on 2D substrates. Matrix compaction and stiffening was shown to be highly localized using confocal and atomic force microscopies, respectively. Myofibroblast phenotype, evaluated by α-smooth muscle actin (α-SMA) immunocytochemistry, significantly increased in matrix regions proximal to bubbles compared to distal regions, irrespective of the addition of exogenous transforming growth factor-β1 (TGF-β1). Introduction of the TGF-β1 receptor inhibitor SB431542 abrogated the proximal enhancement. This approach providing spatiotemporal control over biophysical signals and resulting cell behavior could aid in better understanding fibrotic disease progression and the development of therapeutic interventions for chronic wounds. Statement of SignificanceHydrogels are used in cell culture to recapitulate both biochemical and biophysical aspects of the native extracellular matrix. Biophysical cues like stiffness can impact cell behavior. However, with conventional hydrogels, there is a limited ability to actively modulate stiffness after polymerization. We have developed an ultrasound-based method of spatiotemporally-controlling mechanical and morphological properties within a composite hydrogel, termed an acoustically-responsive scaffold (ARS). Upon exposure to ultrasound, bubbles are non-thermally generated within the fibrin matrix of an ARS, thereby locally compacting and stiffening the matrix. We demonstrate how ARSs control the differentiation of fibroblasts into myofibroblasts in 2D. This approach could assist with the study of fibrosis and the development of therapies for chronic wounds.

Ultrafast dynamics of acoustic vaporization and payload release of phase-shift emulsions

Acoustic droplet vaporization (ADV) is the phase-transitioning of perfluorocarbon emulsions via focused ultrasound. ADV has been utilized in many biomedical applications. For drug delivery and tissue regeneration, we developed composite hydrogel scaffolds, termed acoustically responsive scaffolds (ARSs), which here contained fluorescently labeled monodispersed emulsions (Ø: 12.3 ± 0.8 μm). Using ultra-high speed microscopy, we investigated the dynamics of ADV (2.5 MHz, P-: 6 MPa, pulse length: 6μs) in ARSs containing emulsions with perfluoropentane, perfluorohexane, and perfluorooctane. Vaporization and growth dynamics were studied in micro- to millisecond time scales at frame rates up to 10 Mfps. During ADV, the generated bubbles expanded 4.8-, 2.8-, and 2-fold greater than the initial diameter of the perfluoropentane, perfluorohexane, and perfluorooctane emulsions, respectively. Stable bubbles formed from ADV in perfluoropentane and perfluorohexane, though growth rates post-ADV were significantly different. Bubbles recondensed within perfluorooctane emulsions, which exhibited a 20% decrease in diameter following five repeated ADV events. Stable or transient bubble formation was further compared with classical nucleation theory predictions. Additionally, we compared the release dynamics of fluorescently labeled payloads from the emulsions. The results provide physical insight enabling the modulation of bubble dynamics with ADV and hence release kinetics, which can be used for therapeutic applications.

Release of basic fibroblast growth factor from acoustically-responsive scaffolds promotes therapeutic angiogenesis in the hind limb ischemia model

Pro-angiogenic growth factors have been studied as potential therapeutics for cardiovascular diseases like critical limb ischemia (CLI). However, the translation of these factors has remained a challenge, in part, due to problems associated with safe and effective delivery. Here, we describe a hydrogel-based delivery system for growth factors where release is modulated by focused ultrasound (FUS), specifically a mechanism termed acoustic droplet vaporization. With these fibrin-based, acoustically-responsive scaffolds (ARSs), release of a growth factor is non-invasively and spatiotemporally-controlled in an on-demand manner using non-thermal FUS. In vitro studies demonstrated sustained release of basic fibroblast growth factor (bFGF) from the ARSs using repeated applications of FUS. In in vivo studies, ARSs containing bFGF were implanted in mice following induction of hind limb ischemia, a preclinical model of CLI. During the 4-week study, mice in the ARS + FUS group longitudinally exhibited significantly more perfusion and less visible necrosis compared to other experimental groups. Additionally, significantly greater angiogenesis and less fibrosis were observed for the ARS + FUS group. Overall, these results highlight a promising, FUS-based method of delivering a pro-angiogenic growth factor for stimulating angiogenesis and reperfusion in a cardiovascular disease model. More broadly, these results could be used to personalize the delivery of therapeutics in different regenerative applications by actively controlling the release of a growth factor.

Spatially-directed angiogenesis using ultrasound-controlled release of basic fibroblast growth factor from acoustically-responsive scaffolds

Vascularization is a critical step following implantation of an engineered tissue construct in order to maintain its viability. The ability to spatially pattern or direct vascularization could be therapeutically beneficial for anastomosis and vessel in-growth. However, acellular and cell-based strategies to stimulate vascularization typically do not afford this control. We have developed an ultrasound-based method of spatially- controlling regenerative processes using acellular, composite hydrogels termed acoustically-responsive scaffolds (ARSs). An ARS consists of a fibrin matrix doped with a phase-shift double emulsion (PSDE). A therapeutic payload, which is initially contained within the PSDE, is released by an ultrasound-mediated process called acoustic droplet vaporization (ADV). During ADV, the perfluorocarbon (PFC) phase within the PSDE is vaporized into a gas bubble. In this study, we generated ex situ four different spatial patterns of ADV within ARSs containing basic fibroblast growth factor (bFGF), which were subcutaneously implanted in mice. The PFC species within the PSDE significantly affected the morphology of the ARS, based on the stability of the gas bubble generated by ADV, which impacted host cell migration. Irrespective of PFC, significantly greater cell proliferation (i.e., up to 2.9-fold) and angiogenesis (i.e., up to 3.7-fold) were observed adjacent to +ADV regions of the ARSs compared to -ADV regions. The morphology of the PSDE, macrophage infiltration, and perfusion in the implant region were also quantified. These results demonstrate that spatially-defined patterns of ADV within an ARS can elicit spatially-defined patterns of angiogenesis. Overall, these finding can be applied to improve strategies for spatially-controlling vascularization. Statement of significanceVascularization is a critical step following implantation of an engineered tissue. The ability to spatially pattern or direct vascularization could be therapeutically beneficial for inosculation and vessel in-growth. However, acellular and cell-based strategies to stimulate vascularization typically do not afford this control. We have developed an ultrasound-based method of spatially-controlling angiogenesis using acellular, composite hydrogels termed acoustically-responsive scaffolds (ARSs). An ARS consists of a fibrin matrix doped with a phase-shift double emulsion (PSDE). An ultrasound-mediated process called acoustic droplet vaporization (ADV) was used to release basic fibroblast growth factor (bFGF), which was initially contained within the PSDE. We demonstrate that spatially-defined patterns of ADV within an ARS can elicit spatially-defined patterns of angiogenesis in vivo. Overall, these finding can improve strategies for spatially-controlling vascularization.

Stable and transient bubble formation in acoustically-responsive scaffolds by acoustic droplet vaporization: theory and application in sequential release

Acoustically-responsive scaffolds (ARSs), which are fibrin hydrogels containing monodispersed perfluorocarbon (PFC) emulsions, respond to ultrasound in an on-demand, spatiotemporally-controlled manner via a mechanism termed acoustic droplet vaporization (ADV). Previously, ADV has been used to control the release of bioactive payloads from ARSs to stimulate regenerative processes. In this study, we used classical nucleation theory (CNT) to predict the nucleation pressure in emulsions of different PFC cores as well as the corresponding condensation pressure of the ADV-generated bubbles. According to CNT, the threshold bubble radii above which ADV-generated bubbles remain stable against condensation were 0.4 µm and 5.2 µm for perfluoropentane (PFP) and perfluorohexane (PFH) bubbles, respectively, while ADV-generated bubbles of any size in perfluorooctane (PFO) condense back to liquid at ambient condition. Additionally, consistent with the CNT findings, stable bubble formation from PFH emulsion was experimentally observed using confocal imaging while PFO emulsion likely underwent repeated vaporization and recondensation during ultrasound pulses. In further experimental studies, we utilized this unique feature of ADV in generating stable or transient bubbles, through tailoring the PFC core and ultrasound parameters (excitation frequency and pulse duration), for sequential delivery of two payloads from PFC emulsions in ARSs. ADV-generated stable bubbles from PFH correlated with complete release of the payload while transient ADV resulted in partial release, where the amount of payload release increased with the number of ultrasound exposure. Overall, these results can be used in developing drug delivery strategies using ARSs.

Time-dependent, microscale mechanical properties of acoustically responsive scaffolds using atomic force microscopy and confocal imaging

Acoustic droplet vaporization (ADV) has been used to modulate delivery of regenerative molecules from acoustically responsive scaffolds (ARSs), which are composite fibrin hydrogels containing payload-carrying, phase-shift emulsions. Here, we studied the micromechanical properties of ARSs including stiffness as well as elastic and shear moduli over 7 days post-ADV. ARSs, containing dextran-loaded perfluorohexane (PFH) emulsions (diameter: 6 mm) embedded in fluorescently labeled fibrin gels, were exposed to acoustic pressures above the ADV threshold (2.2 ± 0.2 MPa ) at 2.5 MHz. The compressive stiffness as well as elastic modulus of fibrin gels (i.e., without emulsions) and ARSs (pre- and post-ADV) were measured via force-spectroscopy and the Hertz model, respectively. The measured stiffness was lowest (1.9 ± 0.5 mN/m) in regions adjacent to the PFH emulsions and highest (16.0 ± 4.4 mN/m) in regions adjacent to the ADV-generated bubbles. Fibrin compaction at the bubble–fibrin interface was observed using time-lapsed confocal imaging and correlated with bubble growth at the time points studied here. In addition, different ADV-generated bubble responses in ARSs will be discussed using time-lapsed confocal imaging. Elucidating the mechanical microenvironment within the ARS could be used to control mechanically induced, cellular processes and further the understanding of ADV-triggered drug delivery for regeneration.

Spatially-directed cell migration in acoustically-responsive scaffolds through the controlled delivery of basic fibroblast growth factor

Hydrogels are commonly used in regenerative medicine for the delivery of growth factors (GFs). The spatial and temporal presentations of GFs are critical for directing regenerative processes, yet conventional hydrogels do not enable such control. We have developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), where release of a GF is non-invasively and spatiotemporally-controlled using focused ultrasound. The ARS consists of a fibrin matrix doped with a GF-loaded, phase-shift emulsion. The GF is released when the ARS is exposed to suprathreshold ultrasound via a mechanism termed acoustic droplet vaporization. In this study, we investigate how different spatial patterns of suprathreshold ultrasound can impact the biological response upon in vivo implantation of an ARS containing basic fibroblast growth factor (bFGF). ARSs were fabricated with either perfluorohexane (bFGF-C6-ARS) or perflurooctane (bFGF-C8-ARS) within the phase-shift emulsion. Ultrasound generated stable bubbles in bFGF-C6-ARS, which inhibited matrix compaction, whereas transiently stable bubbles were generated in bFGF-C8-ARS, which decreased in height by 44% within one day of implantation. The rate of bFGF release and distance of host cell migration were up to 6.8-fold and 8.1-fold greater, respectively, in bFGF-C8-ARS versus bFGF-C6-ARS. Ultrasound increased the formation of macropores within the fibrin matrix of bFGF-C8-ARS by 2.7-fold. These results demonstrate that spatially patterning suprathreshold ultrasound within bFGF-C8-ARS can be used to elicit a spatially-directed response from the host. Overall, these findings can be used in developing strategies to spatially pattern regenerative processes. Statement of SignificanceHydrogels are commonly used in regenerative medicine for the delivery of growth factors (GFs). The spatial and temporal presentations of GFs are critical for directing regenerative processes, yet conventional hydrogels do not enable such control. We have developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), where GF release is non-invasively and spatiotemporally-controlled using focused ultrasound. The ARS consists of a fibrin matrix doped with a phase-shift emulsion loaded with GF, which is released when the ARS is exposed to ultrasound. In this in vivo study, we demonstrate that spatially patterning ultrasound within an ARS containing basic fibroblast growth factor (bFGF) can elicit a spatially-directed response from the host. Overall, these findings can be used in developing strategies to spatially pattern regenerative processes.

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

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: 6μm) compared to the -SWF condition, both at sub- and suprathreshold excitation pressures. At 2.5MHz and 4MPa 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 3.25MHz (day 0) and 8.6MHz (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.

Spatiotemporal control of micromechanics and microstructure in acoustically-responsive scaffolds using acoustic droplet vaporization.

Acoustically-responsive scaffolds (ARSs), which are composite fibrin hydrogels, have been used to deliver regenerative molecules. ARSs respond to ultrasound in an on-demand, spatiotemporally-controlled manner via a mechanism termed acoustic droplet vaporization (ADV). Here, we study the ADV-induced, time-dependent micromechanical and microstructural changes to the fibrin matrix in ARSs using confocal fluorescence microscopy as well as atomic force microscopy. ARSs, containing phase-shift double emulsion (PSDE, mean diameter: 6.3 μm), were exposed to focused ultrasound to generate ADV - the phase transitioning of the PSDE into gas bubbles. As a result of ADV-induced mechanical strain, localized restructuring of fibrin occurred at the bubble-fibrin interface, leading to formation of locally denser regions. ADV-generated bubbles significantly reduced fibrin pore size and quantity within the ARS. Two types of ADV-generated bubble responses were observed in ARSs: super-shelled spherical bubbles, with a growth rate of 31 μm per day in diameter, as well as fluid-filled macropores, possibly as a result of acoustically-driven microjetting. Due to the strain stiffening behavior of fibrin, ADV induced a 4-fold increase in stiffness in regions of the ARS proximal to the ADV-generated bubble versus distal regions. These results highlight that the mechanical and structural microenvironment within an ARS can be spatiotemporally modulated using ultrasound, which could be used to control cellular processes and further the understanding of ADV-triggered drug delivery for regenerative applications.

Standing wave assisted acoustic droplet vaporization for dual payload release from acoustically responsive scaffolds

Ultrasound standing waves have been utilized for many biomedical applications. Here, we demonstrate how standing waves can enhance drug release using acoustic droplet vaporization (ADV), which is the phase-transitioning of perfluorocarbon (PFC) emulsions via ultrasound. These experiments utilized acoustically-responsive scaffolds (ARSs), which are composite fibrin hydrogels containing payload-carrying, monodispersed PFC emulsions. Single- and bi-layer ARSs were generated with dextran-loaded emulsions (diameter: 6 mm) containing either perfluorohexane, or perfluorooctane in each layer. First, we studied the influence of standing waves on payload release from single-layer ARSs. At 4 MPa peak rarefactional pressure, elevated amplitudes due to constructive superposition in the standing wave field enhanced payload release up to 35% at 2.5 MHz in a seven-day longitudinal study. Second, the effect of standing waves was combined with the frequency-dependent ADV to enhance dual payload release from bi-layer ARSs. We demonstrated the sequential release of two dextran payloads from ARSs, which were, respectively, contained within each emulsion, using temporally staggered ADV at 3 MHz (day 0) and 8.8 MHz (day 4). These results will also be discussed in the context of practical strategies for achieving similar conditions for in vivo applicationsUltrasound standing waves have been utilized for many biomedical applications. Here, we demonstrate how standing waves can enhance drug release using acoustic droplet vaporization (ADV), which is the phase-transitioning of perfluorocarbon (PFC) emulsions via ultrasound. These experiments utilized acoustically-responsive scaffolds (ARSs), which are composite fibrin hydrogels containing payload-carrying, monodispersed PFC emulsions. Single- and bi-layer ARSs were generated with dextran-loaded emulsions (diameter: 6 mm) containing either perfluorohexane, or perfluorooctane in each layer. First, we studied the influence of standing waves on payload release from single-layer ARSs. At 4 MPa peak rarefactional pressure, elevated amplitudes due to constructive superposition in the standing wave field enhanced payload release up to 35% at 2.5 MHz in a seven-day longitudinal study. Second, the effect of standing waves was combined with the frequency-dependent ADV to enhance dual payload release from bi-layer ARSs. ...

Acoustic Droplet Vaporization in Acoustically Responsive Scaffolds: Effects of Frequency of Excitation, Volume Fraction and Threshold Determination Method

Ultrasound-induced vaporization of liquid perfluorocarbon (PFC) droplets into microbubbles, termed acoustic droplet vaporization (ADV), has potential therapeutic and diagnostic applications. Recently, we demonstrated how ADV—a threshold-based phenomenon—can modulate the release of biomolecules from composite hydrogels, thereby stimulating regenerative processes, such as angiogenesis. These composite hydrogels, called acoustically responsive scaffolds (ARSs), consist of monodispersed, micron size PFC emulsions embedded within a fibrin matrix. This study investigated the effects of frequency of excitation (2.25, 5, 7.5 and 10 MHz) and volume fraction (0.05%, 0.2% and 1% [v/v]) of monodispersed, double emulsions in the ARSs on the ADV threshold. We determined and compared the ADV thresholds via acoustic methods, including active detection, passive detection and attenuation, as well as an echogenicity-based method using B-mode imaging. The ADV threshold determined via these four techniques showed an increasing trend with frequency of excitation. Further analysis of the wave propagation showed that the amplitudes of high frequency harmonics were diminished in ARSs with high volume fractions of emulsion. The ADV threshold inversely correlated with the volume fraction of emulsion at the lowest excitation frequency. However, at higher frequencies, possibly due to the high acoustic reflectivity of the PFC emulsions, the ADV threshold correlated directly with the volume fraction of the emulsion. Additionally, the ADV efficiency correlated with the supra-threshold acoustic pressure. Overall, these results elucidate fundamental acoustic properties of the ARSs, which can be used in future applications.

Parametric Study of Acoustic Droplet Vaporization Thresholds and Payload Release From Acoustically-Responsive Scaffolds

Hydrogels are commonly used for the delivery of bioactive molecules, especially growth factors and cytokines capable of stimulating tissue regeneration. Regenerative processes are regulated by the concentrations and spatiotemporal presentations of these molecules. With conventional hydrogels, these critical delivery parameters cannot be actively modulated after implantation. We have developed composite hydrogel scaffolds where payload release is non-invasively modulated, in an on-demand manner, using ultrasound (US). These acoustically-responsive scaffolds (ARSs) consist of a fibrin matrix doped with a payload-carrying, perfluorocarbon (PFC) double emulsion. Previously, acoustic droplet vaporization (ADV) was used to trigger release of a pro-angiogenic growth factor, encapsulated in the ARS, which stimulated blood vessel formation in vivo. In the present study, we assess how characteristics of the monodispersed emulsion, fibrin matrix, and US impact ADV thresholds and the release efficiency of a dextran payload. ADV thresholds increased with the molecular weight of the PFC in the emulsion and inversely with the volume fraction of emulsion in the ARS. Payload release from ARSs with perfluoroheptane (C7) or perfluorooctane (C8) emulsions was dependent on the number of z-planes of US used to generate ADV and inversely dependent on the lateral spacing. Conversely, release from ARSs with perfluoropentane (C5) or perfluorohexane (C6) emulsions was less dependent on these US exposure parameters. After ADV, payload diffusion decreased significantly in ARSs with C5 or C6 emulsions compared with ARSs with C7 or C8 emulsions. The expansion of the ARS after ADV decreased with the molecular weight of the PFC. Non-selective release increased with the molecular weight of the PFC and thrombin concentration. Overall, these findings can be used for optimization of ARS properties and US parameters in future therapeutic applications.

Sequential Payload Release from Acoustically-Responsive Scaffolds Using Focused Ultrasound

Regenerative processes, such as angiogenesis and osteogenesis, often require multiple growth factors with distinct spatiotemporal patterns and expression sequences. Within tissue engineering, hydrogel scaffolds are commonly used for exogenous growth factor delivery. However, direct incorporation of growth factors within conventional hydrogels does not afford spatiotemporally controlled delivery because release is governed by passive mechanisms that cannot be actively controlled after the scaffold is implanted. We have developed acoustically-responsive scaffolds (ARSs), which are fibrin scaffolds doped with payload-containing, sonosensitive emulsions. Payload release from ARSs can be controlled non-invasively and on demand using focused, megahertz-range ultrasound. In the in vitro study described here, we developed and characterized ARSs that enable sequential release of two surrogate, fluorescent payloads using consecutive ultrasound exposures at different acoustic pressures. ARSs were generated with various combinations and volume fractions of perfluoropentane, perfluorohexane, and perfluoroheptane emulsions. Acoustic droplet vaporization and inertial cavitation thresholds correlated with the boiling point/molecular weight of the perfluorocarbon while payload release correlated inversely. Payload release was longitudinally measured and observed to follow a sigmoidal trend versus acoustic pressure. Perfluoropentane and perfluorohexane emulsions were stabilized when incorporated into ARSs with perfluoroheptane emulsion. These results highlight the potential of using ARSs for sequential, dual-payload release for tissue regeneration.

Controlled release of basic fibroblast growth factor for angiogenesis using acoustically-responsive scaffolds.

The clinical translation of pro-angiogenic growth factors for treatment of vascular disease has remained a challenge due to safety and efficacy concerns. Various approaches have been used to design spatiotemporally-controlled delivery systems for growth factors in order to recapitulate aspects of endogenous signaling and thus assist in translation. We have developed acoustically-responsive scaffolds (ARSs), which are fibrin scaffolds doped with a payload-containing, sonosensitive emulsion. Payload release can be controlled non-invasively and in an on-demand manner using focused, megahertz-range ultrasound (US). In this study, we investigate the invitro and invivo release from ARSs containing basic fibroblast growth factor (bFGF) encapsulated in monodispersed emulsions. Emulsions were generated in a two-step process utilizing a microfluidic device with a flow focusing geometry. At 2.5MHz, controlled release of bFGF was observed for US pressures above 2.2±0.2MPa peak rarefactional pressure. Superthreshold US yielded a 12.6-fold increase in bFGF release invitro. The bioactivity of the released bFGF was also characterized. When implanted subcutaneously in mice, ARSs exposed to superthreshold US displayed up to 3.3-fold and 1.7-fold greater perfusion and blood vessel density, respectively, than ARSs without US exposure. Scaffold degradation was not impacted by US. These results highlight the utility of ARSs in both basic and applied studies of therapeutic angiogenesis.

WE‐AB‐BRA‐03: Non‐Invasive Controlled Release from Implantable Hydrogel Scaffolds Using Ultrasound

Purpose:To control release of a model payload in acoustically responsive scaffolds (ARSs) using focused ultrasound (FUS).Methods:Fluorescently‐labeled dextran (10 kDa) was encapsulated in sonosensitive perfluorocarbon (C6F14 or C5F12) double emulsions (mean diameter: 2.9±0.1 µm). For in vitro release studies, 0.5 mL ARSs (10 mg/mL fibrin, 1% (v/v) emulsion) were polymerized in 24 well plates and covered with 0.5 mL medium. Starting one day after polymerization, ARSs were exposed to FUS (2.5 MHz, Pr = 8 MPa, 13 cycles, 100 Hz PRF) for 2 min daily. The amount of dextran released into the media was quantified. For in vivo studies, 0.25 mL ARSs were prepared as described previously and injected subcutaneously in the lower back of BALB/c mice. After polymerization, a subset of the implanted ARSs were exposed to FUS (as previously described). Animals were imaged longitudinally using a fluorescence imaging system to quantify the amount of dextran released from the ARSs.Results:In vitro: Over 6 days, +FUS displayed an 8.2‐fold increase in dextran release compared to −FUS (−FUS: 2.7±0.6%; +FUS: 22.2±3.0%) for C6F14 ARSs, and a 6.7‐fold increase (−FUS: 5.0±0.8%; +FUS: 38.5±1.6%) for C5F12:C6F14 ARSs. In vivo: +FUS displayed statistically greater dextran release compared to −FUS one day after implantation for C5F12:C6F14 ARSs (−FUS: 55.1±1.5%; +FUS: 74.1±2.2%) and three days after implantation for C6F14 ARSs (−FUS: 1.4±6.5%; +FUS: 30.4±5.4%).Conclusion:FUS enables non‐invasive control of payload release from an ARS, which could benefit growth factor delivery for tissue regeneration. ARS are versatile due to their tunability (i.e. stiffness, emulsion composition, FUS pressure, FUS frequency, etc.) and can be modified to for optimal payload release. Future work will optimize ARS formulations for in vivo use to minimize payload release in the absence of FUS.This work was supported by NIH Grant R21 AR065010 (M.L. Fabiilli) and the Basic Radiologic Sciences Innovative Research Award (M.L. Fabiilli). A. Moncion is supported by the National Science Foundation Graduate Student Research Fellowship (Grant DGE 1256260).

Design and Characterization of Fibrin-Based Acoustically Responsive Scaffolds for Tissue Engineering Applications

Hydrogel scaffolds are used in tissue engineering as a delivery vehicle for regenerative growth factors. Spatiotemporal patterns of growth factor signaling are critical for tissue regeneration, yet most scaffolds afford limited control of growth factor release, especially after implantation. We previously found that acoustic droplet vaporization can control growth factor release from a fibrin scaffold doped with a perfluorocarbon emulsion. This study investigates properties of the acoustically responsive scaffold (ARS) critical for further translation. At 2.5 MHz, acoustic droplet vaporization and inertial cavitation thresholds ranged from 1.5 to 3.0 MPa and from 2.0 to 7.0 MPa peak rarefactional pressure, respectively, for ARSs of varying composition. Viability of C3H/10T1/2 cells, encapsulated in the ARS, did not decrease significantly for pressures below 4 MPa. ARSs with perfluorohexane emulsions displayed higher stability versus those with perfluoropentane emulsions, while surrogate payload release was minimal without ultrasound. These results enable the selection of ARS compositions and acoustic parameters needed for optimized spatiotemporally controlled release.