Core-shell microparticles for protein sequestration and controlled release of a protein-laden core

Abstract

Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation.

Statement of significance: Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic core-shell heparin-PEG microparticles presented here overcome this limitation by sequestering proteins through a PEG-based shell onto a protein-protective heparin core, temporarily isolating bound proteins from the cellular microenvironment, and re-delivering proteins only after degradation of the PEG-based shell. Thus, these core-shell microparticles have potential to be a novel tool to harness and isolate proteins produced in the cellular environment and then control when proteins are re-introduced for the most effective tissue regeneration and repair.

Keywords: Controlled release; Core-shell microparticles; Heparin; Hydrolytically degradable; Protein delivery.

Figure 1

(A) Core-shell MPs were formed via a water-in-oil emulsion. (B) Phase images of core-shell MPs. Arrows and arrowheads indicate the PEG-based shell and heparin core, respectively. (C) Orthogonal view from 3D confocal image stacks confirmed encapsulation of heparin core (red, arrow) into PEG-based shell (green) (scale bar = 25 µm). (D) Histogram of size distribution for core-shell MPs with 1 mg heparin MPs. The average core-shell MP diameter was 58±28 µm and the median 55 µm.

Figure 2

The heparin content in core-shell MP is correlated to MP size and mass of heparin MPs in the precursor PEG solution. (A) Number of heparin MPs correlated linearly with core-shell MP cross-sectional area for five masses of heparin tested. Note axes are different in 0.75 and 1 mg groups due to increases in MP size (n=3 batches of MPs for each mass). (B) Representative images of MPs fabricated with 1 mg of heparin ranging from 35–125 µm in diameter, noted above each image and pointed out by white arrows if more than one MP/image (PEG in green, heparin in red ; scale bar = 25 µm). (C) The ratio of heparin MPs/core-shell size increases as the mass of heparin MPs in precursor PEG phase is increased (n=3 batches of MPs for each mass). (D) Representative images of 0.1, 0.5, and 1 mg heparin MP encapsulated in PEG shell (scale bar = 25 µm).

Figure 3

Degradation of PEG-based shell and release of heparin MP core from core-shell MPs. Core-shell MPs are present through day 5 (days 1–5, arrows), at which point they begin to degrade and release heparin MPs (days 5 and 7, arrowheads). Core-shell MPs are fully degraded by day 7.

Figure 4

Degradable (Deg) core-shell MPs modulate loaded heparin MP delivery to cells. (A) Experimental set-up for pre-fabrication loaded core-shell MPs. (B) Cumulative percent released of loaded BMP-2 for deg core-shell, core-shell, and heparin MP groups (&=significantly different from heparin MP group, p<0.05, n≥3). (C) Normalized ALP activity for deg core-shell, core-shell, and soluble BMP-2 groups (non-loaded groups showed no signal; *=significantly different from core-shell MP group, p<0.05, n=4).

Figure 5

Core-shell MPs temporally modulate protein sequestration. Protein pull-down studies with growth factors (A) SDF-1α and (B) BMP-2. Graphs show percent of protein remaining in solution (i.e. percentage of protein not sequestered by MPs) normalized to soluble protein controls. (*=significantly different from heparin MP group, **=significantly different from degradable core-shell; p<0.05, n≥3).

Figure 6

Post-fabrication loading allows for delivery of loaded heparin MPs. (A) Experimental set-up for post-fabrication loaded MPs. (B) Post-fabrication loading resulted in nearly 100% loading in deg core-shell MP group and less than 10% loading in deg PEG-based MP group. (C) Cumulative mass of BMP-2 released for deg core-shell and deg PEG-based MPs for seven days. (D) Normalized ALP activity for deg core-shell MP, deg PEG-based MP, and soluble groups. “ND” indicates not detectable (non-loaded groups showed no signal) (*=Significantly different from deg core-shell group; p<0.05, n=4).