Direct delivery of functional proteins and enzymes to the cytosol using nanoparticle-stabilized nanocapsules

Abstract

Intracellular protein delivery is an important tool for both therapeutic and fundamental applications. Effective protein delivery faces two major challenges: efficient cellular uptake and avoiding endosomal sequestration. We report here a general strategy for direct delivery of functional proteins to the cytosol using nanoparticle-stabilized capsules (NPSCs). These NPSCs are formed and stabilized through supramolecular interactions between the nanoparticle, the protein cargo, and the fatty acid capsule interior. The NPSCs are ~130 nm in diameter and feature low toxicity and excellent stability in serum. The effectiveness of these NPSCs as therapeutic protein carriers was demonstrated through the delivery of fully functional caspase-3 to HeLa cells with concomitant apoptosis. Analogous delivery of green fluorescent protein (GFP) confirmed cytosolic delivery as well as intracellular targeting of the delivered protein, demonstrating the utility of the system for both therapeutic and imaging applications.

Figure 1

Design and preparation of nanoparticle-stabilized capsules (NPSCs). (a) Schematic showing the preparation of the protein-NPSC complex containing caspase-3 or GFP and proposed delivery mechanism. The oil was a 1:1 mixture of linoleic acid (LA) and decanoic acid (DA). (b) TEM image of the dried GFP-NPSC. (c) Dynamic light scattering (DLS) histogram of GFP-NPSCs indicating an average diameter of 130 ± 40 nm.

Figure 2

Delivery of caspase-3 into HeLa cells. Cells were incubated for 1 h with (a) CASP3-NPSC, (b) NPSC without CASP3, and (c) only CASP3 without NPSC. Subsequently, cells were stained using Yopro-1 (green fluorescence) and 7-AAD (red fluorescence) for 30 min, and the overlapped images are presented as apoptotic. Individual channel images are shown in Figure S5. (d) Apoptosis ratios of the cells after CASP3 delivery. Scale bars: 100 μm; the error bars represent the standard deviations of three parallel measurements.

Figure 3

Delivery of GFP into HeLa cells. (a) Confocal image showing GFP delivery into HeLa cells by NPSCs. (b) Confocal images showing the colocalization of delivered GFP with expressed mCherry in HeLa cell. (c) Flow cytometry results of HeLa cells treated with GFP-NPSCs (red), or GFP alone (blue) for 2 h, using untreated HeLa cells as the control (black). Scale bars: 20 μm.

Figure 4

Live cell imaging of rapid GFP release into the cytosol of HeLa cell by NPSCs. “0 min” label represents the starting point of release. The arrow indicates a GFP-NPSC at the cell membrane prior to delivery of payload. Scale bar: 20 μm.

Figure 5

Peroxisome targeting in HeLa cells transfected with RFP-PTS1 plasmid. (a) Colocalization of GFP-PTS1 fusion protein with the peroxisomal indicator (RFP-PTS1) expressed by the cell. (b) No colocalization of GFP was observed without PTS1 motif. Scale bars: 20 μm.