Sequential release of nanoparticle payloads from ultrasonically burstable capsules

Abstract

In many biomedical contexts ranging from chemotherapy to tissue engineering, it is beneficial to sequentially present bioactive payloads. Explicit control over the timing and dose of these presentations is highly desirable. Here, we present a capsule-based delivery system capable of rapidly releasing multiple payloads in response to ultrasonic signals. In vitro, these alginate capsules exhibited excellent payload retention for up to 1 week when unstimulated and delivered their entire payloads when ultrasonically stimulated for 10-100 s. Shorter exposures (10 s) were required to trigger delivery from capsules embedded in hydrogels placed in a tissue model and did not result in tissue heating or death of encapsulated cells. Different types of capsules were tuned to rupture in response to different ultrasonic stimuli, thus permitting the sequential, on-demand delivery of nanoparticle payloads. As a proof of concept, gold nanoparticles were decorated with bone morphogenetic protein-2 to demonstrate the potential bioactivity of nanoparticle payloads. These nanoparticles were not cytotoxic and induced an osteogenic response in mouse mesenchymal stem cells. This system may enable researchers and physicians to remotely regulate the timing, dose, and sequence of drug delivery on-demand, with a wide range of clinical applications ranging from tissue engineering to cancer treatment.

Keywords: Biomaterials; Controlled release; Drug delivery; Ultrasound response.

Fig. 1

Alginate capsules can be fabricated to rupture in response to ultrasound. (A) Schematic of the capsule fabrication process: (i) droplets containing divalent cations, sucrose, and nanoparticle payloads are added drop-wise to an alginate bath, (ii) once submerged, these droplets release divalent cations which interact with the alginate, (iii) eventually forming an ionically cross-linked capsule wall. (iv) A representative microscopic image of a capsule loaded with iron oxide particles formed using 50 mM CaCl₂. (B) Histograms of capsule diameter and wall thickness (i,ii) distributions and (iii) comparisons of mean values ± standard deviations (N = 8). (C) Images of 10 mL PBS solutions containing iron-oxide-loaded capsules when subjected to the indicated ultrasonic signals for 5 seconds. These experiments were conducted in glass scintillation vials with the 13-mm ultrasound probe placed 2 cm above the capsules.

Fig. 2

Capsules retain their nanoparticle payloads for prolonged periods and can be ultrasonically triggered to release their payloads. (A) Percent release over time from gold-nanoparticle-loaded capsules (inset) that were exposed to ultrasound on day 7 for 100 s at 20% ultrasound. (B) For the same experiment as presented in part A, the rate of nanoparticle release over time. In parts A and B, the red shaded region at day 7 indicates the time when capsules were exposed to ultrasound. (C) The percent release as a function of the duration of ultrasonic exposure for capsules exposed to 20% (black), 40% (blue) and 80% (red) amplitudes. Note that the x-axis is log-scale. (D) Percent release as a function of the ultrasound’s duration when exposed to 20% ultrasound for capsules immediately after fabrication (solid curve) compared to capsules that sat in PBS for a week (dashed curve). For parts A–D, N = 4. In each experiment (A–D), capsules were placed in 50 mL tubes containing 5 mL of PBS with the sonicator’s probe being centered azimuthally and placed 2 cm above the bottom of the tube.

Fig. 3

Capsules can be engineered with varying strength and susceptibility to ultrasonic rupture to enable sequentially triggered payload deliveries. (A) Microscopic images of (i) weak capsules made by using 50 mM CaCl₂ and (ii) strong capsules made by using 100 mM BaCl₂. Yellow arrows draw attention to the capsule wall boundaries. (B) Comparison of weak (red) and strong (blue) capsules’ inner diameter (ID), capsule wall thickness, and outer diameter (OD). N = 17–24. (C) Applied force as capsules were compressed at 2 mm/min until failure (indicated by ‘x’) for weak (red) and strong (blue) capsules. Inset: mean ± standard deviations of the forces required to rupture weak and strong capsules under compression. N = 8. (D) The percent of gold nanoparticles released from strong and weak capsules as a function of ultrasonic exposure duration when exposed to (i) 20% and (ii) 80% ultrasound amplitudes. (E) Percent gold nanoparticle release over time for when both weak and strong capsules were exposed to 200 s of ultrasound on day 7 at 20% amplitude followed by 100 s of ultrasound five minutes later at 80% amplitude. For parts D and E, N = 4. (F) Images of weak (red) and strong (blue) capsules as they were exposed to ultrasound at 80% amplitude for long enough to rupture the weak capsules (10 s), followed by a pause (“no ultrasound”), and then an additional ultrasound to rupture the strong capsules (for an 100 additional seconds). Weak and strong capsules were loaded with red and blue food coloring, respectively.

Fig. 4

The gold nanoparticles loaded into these ultrasonically responsive capsules can be endowed with osteogenic bioactivity. (A) (i) Images of live/dead stained mMSCs after exposure to 211.2 μg/mL of gold nanoparticles in DMEM for 7 days compared to a control with no nanoparticles. (ii) Quantitation of mMSC viability when exposed to gold nanoparticles and controls (N = 4). (B) (i) BMP-2 includes several cysteine residues (ii) which contain disulfide bonds which can bind to gold substrates. (C) Detected BMP-2 concentrations when assaying the indicated concentrations of undecorated gold nanoparticles (gray) and BMP-2-decorated particles (black). n.d. indicates that no protein was detected using ELISA. The 211.2 μg/mL condition saturated the assay when using BMP-2-decorated nanoparticles. (D) Relative ALP activity (normalized to osteogenically supplemented media) for mMSCs after 7 days when exposed to the following: moving from left to right, normal culture media (DMEM), osteogenic supplemented cell media (osteo-DMEM), osteo-DMEM with 10 and 300 ng/mL of BMP-2, osteo-DMEM with 2.62 μg/mL of PEG-decorated gold nanoparticles, and osteo-DMEM with 2. 62 μg/mL of BMP-2-decorated gold nanoparticles (estimated to be equivalent to 300 ng/mL of BMP-2 when using ELISA) (N = 3–4). * indicates statistically significant differences when compared to both DMEM and osteo-DMEM conditions (p < 0.01) and † indicates statistically significant differences when compared to the 10 ng/mL BMP-2 condition (p < 0.05).

Fig. 5

Capsules can be ruptured when integrated into hydrogels and placed in a chicken carcass tissue model using ultrasonic signals that do not result in tissue heating or reductions in cell viability. (A) Images of iron-oxide-loaded capsules integrated into 2 wt % alginate hydrogels that are cross-linked with 5 mM calcium sulfate before, during, and after ultrasonic stimulation when implanted under the skin of 37°C chicken carcass. The time over the course of each experiment moves from left to right. Note that gels containing only weak capsules (top row) were not stimulated at 80% ultrasound since all capsules ruptured using 20% ultrasound. Therefore, an image was not acquired for the 80% case. (B) Tissue temperature before and after exposure to 10 seconds of ultrasound at 80% amplitude. (C) Cell viably (black, left axis) and cell count (grey, right axis) of mMSCs encapsulated in 2 wt % alginate hydrogels that are cross-linked with 5 mM calcium sulfate and exposed to 10 seconds of ultrasound at 80% amplitude (N = 4).