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

Abstract

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.

Introduction

Critical limb ischemia (CLI), an advanced stage of peripheral artery disease (PAD), is the third leading cause of atherosclerotic morbidity [1]. In CLI, arterial blockages reduce blood flow in the extremities, thus leading to chronic pain, numbness, limited mobility and tissue death. The gold standard in CLI treatment is surgical or endovascular revascularization. However, even with intervention, the prognosis for CLI patients is grim. Rates of restenosis can exceed 80% [2] and 29% of patients will either die or undergo a major amputation within one year of diagnosis [3]. Furthermore, 25% of CLI patients are ineligible for revascularization therapy because of existing co-morbidities [4]. Due to these limited options, experimental treatments have sought to improve tissue perfusion by stimulating the growth of new blood vessels to circumvent occluded vessels. One approach that has been evaluated in both preclinical [[5], [6], [7]] and clinical trials [8,9] of PAD/CLI is the use of exogenous, pro-angiogenic growth factors (GFs) such as basic fibroblast growth factor (bFGF). However, conventional administration routes (e.g., intramuscular or intravascular injections) are ineffective for GFs, which have contributed to translational challenges [10].

Hydrogels are widely used within regenerative medicine to deliver GFs [11,12]. These water-laden matrices have been shown to increase the in vivo half-lives of GFs and facilitate angiogenesis. Release of a GF from a hydrogel is dominated by mechanisms like diffusion and matrix degradation [13]. As such, the ability to spatially and/or temporally control GF release from a conventional hydrogel is limited. Comparatively, endogenous pro-angiogenic GFs are expressed in spatiotemporally-regulated patterns during regeneration [14]. Upon implantation of a conventional hydrogel, the kinetics of GF release cannot be actively altered, which hinders the ability to personalize GF therapy based on patient response.

We have developed a composite hydrogel that enables control of GF release using therapeutic, focused US (FUS). Globally, FUS is used clinically in both diagnostic and therapeutic applications [15]. FUS can be applied non-invasively, focused with sub-millimeter precision, and delivered in a spatiotemporally-defined manner to deeply located tissues. Our composite hydrogel, termed an acoustically-responsive scaffold (ARS), consists of a fibrin matrix containing a GF-loaded, phase-shift double emulsion (Fig. 1A) [16,17]. Fibrin, a component of the provisional matrix during wound healing, can facilitate angiogenesis and is approved by the United States Food and Drug Administration as a hemostatic agent [18]. Fibrin is enzymatically degraded within the body by plasmin and matrix metalloproteinases [19]. The phase-shift double emulsion has a water-in-perfluorocarbon (PFC)-in-water (W₁/PFC/W₂) structure. A water-soluble payload, like a GF, is contained within the innermost W₁ phase. The PFC phase, which surrounds the W₁ droplets, is highly hydrophobic, thereby acting as a diffusion barrier for the encapsulated GF. PFCs, like perfluoro-n-alkanes, are used in biomedical applications since they are inert, non-metabolizable, and excreted via exhalation [20]. When exposed to FUS, the PFC phase is vaporized due to the tensile (i.e., rarefactional) part of the acoustic wave. This mechanism is known as acoustic droplet vaporization (ADV) and is a threshold-dependent process [21,22]; thus ADV occurs when the applied acoustic pressure (P) is greater than the threshold pressure for ADV (P_ADV). The double emulsion morphology of the phase-shift emulsion is disrupted by ADV, which releases the encapsulated payload [23]. Thus, ADV enables on-demand release of a GF from an ARS.

Previously, FUS-modulated release of bFGF from an ARS stimulated endothelial network formation in an in vitro co-culture model [24]. Additionally, when ARSs were implanted subcutaneously, FUS-stimulated release of bFGF caused increases in angiogenesis and perfusion [25]. Spatial patterning of ADV in an ARS, and hence bFGF release, was also shown to elicit spatially-directed, host cell migration following in vivo implantation [26].

In prior studies, ARSs were implanted in non-ischemic tissues. Here, we investigate for the first time the impact of bFGF release from an ARS in a murine model of hind limb ischemia (HLI) [27] - a widely-used, preclinical model of CLI (Fig. 1B). bFGF was encapsulated in a phase-shift double emulsion and subsequently incorporated within an ARS. The effect of single versus repeated FUS exposure on bFGF release was initially evaluated in vitro. Subsequently, ARSs were implanted in mice following induction of HLI and ischemia as well as perfusion were longitudinally monitored during the 28-day study. Angiogenesis, fibrosis, and macrophage infiltration were assessed using histological and immunohistochemical staining.