Nanoparticles engineered to bind cellular motors for efficient delivery

Abstract

Background: Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications.

Results: Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections.

Conclusions: The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Keywords: Biomimetic synthetic peptides; Drug delivery; Dynein; Microtubule motors; Nanoparticles.

Fig. 1

a Scheme of NPs synthesis (Au@tiopronin) and functionalization with dynein-binding peptides (DBP) and PEG. b TEM image of Au@tiopronin. Scale bar: 20 nm

Fig. 2

Cellular distribution of NPs. a, d Mean fluorescence intensity (MFI) of intracellular Au@tiopronin modified with DynPro (Au@DynPro) or IntCt (Au@IntCt) after 1 h incubation with Vero cells (a) and other cell lines (d). Differences in MFI between Au@DBPs and Au@IntCt (control) were statistically significant p value of 0.001 (α = 0.05). In contrast, differences among Au@DBPs were not significant with a p value of 0.3 (α = 0.05; not shown). b Representative confocal images of Vero cells incubated with Au@DynPro or c Au@IntCt. e–h Representative confocal images of SK-N-MC (e), HeLa (f), 293T (g) and MDCK cells incubated with Au@DynPro. Scale bar: 10 µm. i Time-dependent accumulation of Au@DynPro at several time points between 20 and 120 min, as indicated. j Fluorescence intensity percentages at increasing doses of Au@DynPro quantified by flow cytometry

Fig. 3

Representative confocal images at low magnification of a Vero cells with Au@DynPro b HEK293T cells with Au@ShortPro and c MDCK cells with Au@TransRb. Representative images showed similar NP dispersion throughout the culture and cell-to-cell transfer of Au@DynPro through short (b) or long (c) projections. d, e Representative time-lapse images of the linear progression of Au@DBP inside the cell and the resulting trajectories (circles). f Comparison with the non-linear movement obtained with control NPs Au@IntCt. Scale bar: 5 µm

Fig. 4

a Accumulation of Au@DynPro at the MTOC near the nucleus (N). b Progression of Au@DynPro towards the MTOC, or c nucleus and projections (arrows) in GFP-tubulin transfected cells. d–i Au@DynPro´s mobility was sensitive to microtubule-depolimerizing drugs. d, g Widespread cellular distribution of Au@DynPro before depolymerizing drug treatment, e Au@DynPro transport blockade and accumulation after 1 h drug incubation with 2.5 µM Nocodazole, and f mobility and dispersion recovery after washing. g Vero cells treated with 10 µM Au@DynPro and then h incubated for 1 h with 0.1 µM actin depolymerizing-drug LatrunculinA. This drug produced cell shrinkage because of actin cytoskeleton collapse that was not recovered after washing (i). However, Au@DynPro transport was still preserved within cells and projections. Scale bar: 10 µm

Fig. 5

a, b Nuclear penetration capacity of Au@DynPro (n: nucleus). c–e Nuclear envelope appeared discontinuous as Au@DynPro shuttle the nucleus in Vero cells transfected with GFP-laminB receptor (c) and GFP-laminB1 (d, e) as shown in equatorial optical sections. Sample images show NPs entering the nucleus and nuclear lamina folding at sites of NPs entry. f Peptides shuttle the nucleus in Vero cells transfected with GFPB23 nucleolin (g) Au@DynPro in their way across the nucleus with visible imprints of their paths (b, arrow). Scale bar: 5 μm