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

Abstract

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 in vitro and in vivo 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 2.5 MHz, controlled release of bFGF was observed for US pressures above 2.2 ± 0.2 MPa peak rarefactional pressure. Superthreshold US yielded a 12.6-fold increase in bFGF release in vitro. 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.

Keywords: Acoustic droplet vaporization; Angiogenesis; Basic fibroblast growth factor; Controlled release; Perfluorocarbon; Ultrasound.

Figure 1

Control of angiogenesis using an ARS. (I) ARSs were polymerized in situ in the subcutaneous space. The fibrin-based ARSs contained bFGF encapsulated within a monodispersed double emulsion. (II) During US exposure, the PFC within the emulsion transitioned from a liquid into a gas, thereby releasing the encapsulated bFGF. (III) The released bFGF stimulated blood vessel growth into the ARS.

Figure 2

(A) An image highlighting the flow focusing geometry of the microfluidic device, including the 14 × 17 μm junction where the monodispersed double emulsions were formed. (B) Confocal microscopy images of the resulting double emulsion at 40× and 100× magnification for the large and inset images, respectively. The images show emulsions that are visually uniform in size. Scale bars: 25 μm (large image) and 2.5 μm (inset)

Figure 3

(A) Volume-weighted size distribution of the monodispersed double emulsion. The mean diameter and coefficient of variance of the emulsion was 13.9 ± 0.04 μm and 4.5%, respectively. (B) The scattered, fundamental frequency (i.e., 2.5 MHz) was passively recorded and used as an indicator of ADV, specifically the presence of bubbles generated in the ARS. (C) At pressures above the ADV threshold, 2.2 ± 0.2 MPa, bubble formation was evident. Additionally, the scattered broadband noise was also recorded and used as an indicator of IC, caused by rapid expansion and collapse of the bubbles formed through ADV at high pressures. The IC threshold was 4.8 ± 1.5 MPa (n=3 for both ADV and IC measurements). (D) Stability of the ARS, specifically the emulsions within the ARS, when placed in a cell culture incubator at 37°C (n=5). Statistically significant differences (p < 0.05) relative to day 0 for n=5 samples are denoted by α.

Figure 4

Images of ARSs after exposure to various US pressures on day 1. ARSs exposed to superthreshold US (i.e. > 2.2 MPa) show bubble formation on day 1, while subthreshold US (i.e. < 2.2 MPa) exposures yield no bubble formation. Scale bar: 8 mm. The generated bubbles increase in size by day 2 due to in-gassing.

Figure 5

US was used to control the release of bioactive, bFGF from an ARS (A) The percent of bFGF released as a function of acoustic pressure where “Daily” indicates US exposure on days 1–7. A delayed release experiment was also performed (i.e., “Delayed”) where US was applied on days 4–7. (B) The bioactivity of the released growth factor was determined by incubating NR-6-R fibroblasts with releasate. Cell proliferation was measured after 44 hours. Releasates obtained at 0 or 2 MPa did not contain enough bFGF to induce cell proliferation. All data is represented as mean ± standard error of the mean for n = 5 ARSs. For (A), statistically significant differences (p < 0.05) are denoted as follows. α: 8 MPa (daily) vs. –US; β: 4 MPa (daily) vs. –US; χ: 8 MPa (daily) vs. 4 MPa (daily); ε: 8 MPa (daily) vs. 8 MPa (delayed); η: 4 MPa (daily) vs. 8 MPa (delayed); δ: 8 MPa (delayed) vs. –US. For (B), statistically significant differences (p < 0.05) are denoted as follows. α: vs. fibrin+bFGF on day 2, β: vs. fibrin+bFGF on day 6.

Figure 6

Longitudinal LASCA images of two mice, each with two implants. The regions of interests (ROIs) were chosen based on the physical location of the implants, and are denoted by colored circles. The left most images are visible images of the mice. For all images, the caudal direction is left. ROI diameter: 0.9 cm.

Figure 7

US increased perfusion and blood vessel growth in the bFGF-loaded ARSs (A) Quantification of LASCA images using ROIs. The greatest change in perfusion was observed on day 7, with ARS+US exhibiting greater perfusion than ARS. The differences between ARS and ARS+US were significant on days 7 and 10. All data is represented as mean ± standard error of the mean for n = 8 ARSs (days 1–7) and n = 4 ARSs (days 10 and 14). Statistically significant differences (p < 0.05) are denoted as follows. β: Fibrin vs. ARS; γ: Fibrin vs. ARS+US; η: Fibrin+bFGF vs. ARS; ε: Fibrin+bFGF vs. ARS+US; δ: ARS vs. ARS+US. (B) Blood vessels were identified using CD31 staining. On both days 7 and 14, the greatest blood vessel density was observed for ARS + US. All data is represented as mean ± standard error of the mean for n = 8 ARSs (days 1–7) and n = 4 ARSs (days 10 and 14). Statistically significant differences (p < 0.05) are denoted as follows. α: Fibrin vs. Fibrin+bFGF; β: Fibrin vs. ARS; γ: Fibrin vs. ARS+US; η: Fibrin+bFGF vs. ARS; ε: Fibrin+bFGF vs. ARS+US; δ: ARS vs. ARS+US.

Figure 8

Degradation of the subcutaneously-implanted ARSs, which contained Alexa Fluor 647-labeled fibrinogen, was longitudinally monitored using a fluorescence, in vivo imaging system. All data is represented as mean ± standard error of the mean for n = 8 ARSs (days 1–7) and n = 4 ARSs (day 14).