Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells

Abstract

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell's membrane and subsequently release surface-bound molecules directly into the cell's cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology's potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

Fig. 1.

Si NWs as a generalized platform for delivering a wide range of biological effectors. (A) and (B) Scanning electron micrographs of vertical Si NWs fabricated via CVD and reactive ion etching, respectively. Scale bar in A and B, 1 μm. (C) and (D) Schematic renderings of cells (Pink) on Si NWs (Green) at early and late stages of penetration, respectively. Molecules of interest (X & Y) are nonspecifically tethered to the Si NW surface via alkoxysilane treatment. Upon penetration and delivery, those molecules can alter the cell’s behavior. (E) and (F) Confocal microscope sections (corresponding to the dashed boxes in C and D) of a HeLa cell in the process of being penetrated. Scale bar in E and F, 10 μm. (E) After 15 minutes, the cell membrane, labeled with DiD, sits atop the tips of the wires that are coated with cystamine and Alexa Fluor 488 SE (as shown in C). (F) Within one hour, the same plane shows a magenta circle, the cell’s circumference, surrounding green-labeled wires, indicating NW penetration into the cell (as shown in D). Regardless of the molecular coating, the majority of cells are penetrated within one hour (Fig. S2C). (G) Scanning electron micrograph of rat hippocampal neurons (false colored Yellow) atop a bed of etched Si NWs (false colored Blue), showing characteristic morphology (taken one day after plating). Scale bar, 10 μm.

Fig. 2.

Si NWs can deliver and codeliver diverse biomolecular species to a broad range of cell types. (A)–(D) Various immortalized and primary cell types expressing fluorescent proteins after being transfected with plasmid DNA via the Si NWs: (A) HeLa S3 cells (pCMV-TurboRFP), (B) human fibroblasts (pCMV-TurboRFP), (C) NPCs (pCMV-mCherry), and (D) primary rat hippocampal neurons (pCMV-dTomato). Scale bar in A–D, 25 μm. (E)–(H) Human fibroblasts transfected with a variety of biochemicals. To verify intracellular delivery, following a 24-h incubation on coated Si NWs, cells were replated on glass coverslips for imaging. Epifluorescence images show > 95% of the cells receive: (E) siRNA (Alexa Fluor 546 labeled AllStar Negative Control siRNA), (F) peptides (rhodamine labeled 9-mer), (G) plasmid DNA (labeled with Label-IT Cy5), and (H) proteins (recombinant TurboRFP-mito). Scale bars in E–H, 50 μm. (I)–(L) Fluorescence images of human fibroblasts cotransfected with two different molecules using Si NWs: (I) two different siRNAs (Alexa 546 labeled siRNA—yellow & Alexa 647 labeled siRNA—magenta), (J) a peptide and an siRNA (rhodamine labeled peptide—yellow & Alexa 647 labeled siRNA—magenta), (K) plasmid DNA and an siRNA (Cy5-labeled TagBFP plasmid DNA—yellow & Alexa 546 labeled siRNA—magenta), and (L) a recombinant protein and an siRNA (recombinant TurboRFP-mito—yellow & Alexa 647 labeled siRNA—magenta). In E to L, to facilitate identification, cell membranes were labeled with fluorescein (Gray). Scale bars in I–L, 25 μm.

Fig. 3.

Mitotic inhibition via patterned Si NW delivery of Ara-C. Here, dissociated NPCs have been plated on a line-patterned Si NW substrate that was blanket coated with the small molecule mitotic inhibitor Ara-C. (A) NPCs plated on a line-patterned Si NW substrate in the absence of Ara-C divide out isotropically from their point of plating. (B) Cell division is selectively stopped as cells enter or cross regions where the Si NWs deliver Ara-C (dashed squares). Scale bars, 100 μm.

Fig. 4.

Gene knockdown via NW-mediated siRNA delivery. (A) Fluorescence image showing patch clamping of a typical rat hippocampal neuron (E18) on Si NWs (8 d after plating). (B) Applied hyper- and depolarizing voltage steps. (C) and (D) Current responses of a neuron transfected with a control siRNA (GNU) and siRNA targeted against sodium channels (Na_v1.X), respectively. Panel C displays fast inward sodium currents and outward delayed-rectifier potassium currents while D only shows the latter. (E) and (F) Immunofluorescence images of rat hippocampal neurons on Si NWs transfected with siRNA for GNU and Na_v1.X respectively. Cells were stained for voltage-gated sodium channel type 1 alpha (Scn1a, Pink) and beta-III-tubulin (Green). (F) shows several cells (dashed circles) with significantly reduced expression of sodium channels compared to E.

Fig. 5.

Apoptosis inhibition by Si NW delivered peptides. (A)–(C) Schematics of apoptosis inhibition via peptide delivery: (A) control culture of HeLa cells on Si NWs, (B) bath application of apoptosis-inducing agents Act-D and TNF-α (Red Squares), and (C) bath application of Act-D and TNF-α with wires coated in the apoptosis-inhibiting peptide Ac-DEVD-CHO (Blue Triangles). (D)–(F) TUNEL assay of HeLa cells on Si NWs. (D)–(F) Dead nuclei (Pink), for the experiments depicted in A–C respectively. (G)–(I) fluorescence images showing the labeled cell membranes (Green) of all cells assayed in D–F respectively. (E) shows bath application of Act-D and TNF-α induced apoptosis (as compared to the control D), while NW-mediated delivery of Ac-DEVD-CHO effectively inhibits the induced apoptosis (F).

Fig. 6.

The NW-delivery platform can be combined with microarray printing to deliver bioactive molecules in a site-specific fashion. (A) Epifluorescence image (4x) of HeLa S3 cells grown on top of a two-molecule microarray (4 d after plating). The array, printed on the NW substrate with a 400 - μm pitch, consists of siRNAs targeted against the intermediate filament vimentin (labeled with Alexa Fluor 546, Pink) and a nuclear histone H1 protein (labeled with Alexa Fluor 488, Green). (B) Confocal image (625 μm × 625 μm) showing the subcellular localization of the siRNAs and histones in the cells. (C) and (D) Confocal zoom-ins of individual spots where siRNAs and histones have been delivered, respectively. (C) Immunostaining for vimentin (Green) and nuclei (Blue) shows that a group of cells receiving siRNAs (Red), outlined in the dotted square, display knocked-down vimentin expression. (D) Cells in the dotted square show histones (Green) intracellularly delivered and subsequently targeted to the cells’ nuclei (Blue). Scale bar in A and B (C and D), 100 (50) μm.