Vertical silicon nanowires as a universal platform for highly efficient delivery of bioactive molecules into living cells
This platform consists of an array of surface-modified vertical silicon nanowires that have been chemically grown or etched on a wafer and to which virtually any kind of bioactive species (drug, protein, nucleic acid, nanoparticles) may be attached by covalent or electrostatic means. When cells are plated on the surface of the array, they are penetrated by the functionalized nanowires, and the bioactive material is thus efficiently delivered into the cytosol of the cells. Since the cells remain viable after penetration, the effects of the delivered material on the cells can be monitored over time.
By optimizing the cell-nanowire interface through the use of novel surface chemistry and the precise control over nanowire density, diameter, and height, investigators in the laboratory of Professor Hongkun Park at Harvard University have created a platform suitable for delivering a wide range of materials (including small molecules, nanoparticles, siRNAs, and proteins) into living cells in a highly efficiently, massively parallel, arrayed fashion. There are three general steps involving to implement the procedure:
1. The nanowires are fabricated by either growing them from a precursor material on the surface of a patterned substrate, or by etching the structures out of a substrate material from the top down.
2. The surface of the nanowires is then chemically treated to facilitate their electrostatic linkage to the compound of interest. (Covalent linkage strategies may also be employed.)
3. The compound of interest is then tethered to the nanowire by pipetting a few uLs of a solution containing the compound atop the nanowires. Microarraying techniques may also be used for the multiplex deposition of an assortment of molecular species. Once the solvent evaporates, the nanowire wafer may be used immediately or stored until needed.
4. Cells are then plated on the nanowire surface. As they settle, they are impaled by the wires (typically within an hour) and the electrostatically linked agent detaches from the wires and is thus released into the cell cytosol.
This single platform has been shown to be able to deliver small molecules, nanoparticles, RNAs, peptides, and proteins, into a number of primary and clonal cell with an efficiency greater than 95%. It should be noted that the Park laboratory has been able to mass-produce the wires on six-inch silicon wafers using a technique that could easily be implemented by any semiconductor foundry. The high-throughput, low-cost production of these silicon wafer arrays using widely available semiconductor processing should enable the wide adoption and rapid commercialization of this technology.
Applications
Existing strategies for delivering exogenous materials into cells (e.g., viral vectors, physio-chemical means, or microinjection) are limited either by the range of chemical and biological species that each can deliver, or by the efficiency or throughput of the delivery. While there have been previous efforts to use nanostructured materials to introduce bioactive species into cells, they too have been inefficient and generally limited to the delivery of genetic material. The potential to deliver a wide range of molecules at sites of one’s choosing lends itself to a broad variety of implementations and applications, including the following:
1. Massive, parallel screening for drug discovery: the cellular effects of many proteins (proteomics), siRNAs (knockdown efficacy), and small molecules (drug/antibiotic resistance) may be assayed simultaneously, either singly or in combinations; by perturbing different elements of a particular cellular pathway, the causal relationships between those elements can be discovered.
2. Cell-based ADME/Tox assays: the concentration-dependent effects of a particular protein, drug, and/or combinations can be studied in cell-based assays.
3. Derivation of induced pluripotent stem (iPS) cells: the platform is suitable for efficiently delivering (and discovering) molecules capable of reprogramming adult cells into iPS cells.
4. Designer cell networks and disease models: the platform can be used to construct complex cellular systems, such as synaptic pairs of differentially perturbed neurons for modeling neurodegenerative diseases.
5. Assays of epigenetic factors: the platform could be used to discover molecules that usefully affect the differentiation and development of any set of cells (e.g., stem cells, iPS cells, or differentiated tissues), and then further used to generate cells of a particular lineage for use in cell-based therapies.
This platform consists of an array of surface-modified vertical silicon nanowires that have been chemically grown or etched on a wafer and to which virtually any kind of bioactive species (drug, protein, nucleic acid, nanoparticles) may be attached by covalent or electrostatic means. When cells are plated on the surface of the array, they are penetrated by the functionalized nanowires, and the bioactive material is thus efficiently delivered into the cytosol of the cells. Since the cells remain viable after penetration, the effects of the delivered material on the cells can be monitored over time.
By optimizing the cell-nanowire interface through the use of novel surface chemistry and the precise control over nanowire density, diameter, and height, investigators in the laboratory of Professor Hongkun Park at Harvard University have created a platform suitable for delivering a wide range of materials (including small molecules, nanoparticles, siRNAs, and proteins) into living cells in a highly efficiently, massively parallel, arrayed fashion. There are three general steps involving to implement the procedure:
1. The nanowires are fabricated by either growing them from a precursor material on the surface of a patterned substrate, or by etching the structures out of a substrate material from the top down.
2. The surface of the nanowires is then chemically treated to facilitate their electrostatic linkage to the compound of interest. (Covalent linkage strategies may also be employed.)
3. The compound of interest is then tethered to the nanowire by pipetting a few uLs of a solution containing the compound atop the nanowires. Microarraying techniques may also be used for the multiplex deposition of an assortment of molecular species. Once the solvent evaporates, the nanowire wafer may be used immediately or stored until needed.
4. Cells are then plated on the nanowire surface. As they settle, they are impaled by the wires (typically within an hour) and the electrostatically linked agent detaches from the wires and is thus released into the cell cytosol.
This single platform has been shown to be able to deliver small molecules, nanoparticles, RNAs, peptides, and proteins, into a number of primary and clonal cell with an efficiency greater than 95%. It should be noted that the Park laboratory has been able to mass-produce the wires on six-inch silicon wafers using a technique that could easily be implemented by any semiconductor foundry. The high-throughput, low-cost production of these silicon wafer arrays using widely available semiconductor processing should enable the wide adoption and rapid commercialization of this technology.
Existing strategies for delivering exogenous materials into cells (e.g., viral vectors, physio-chemical means, or microinjection) are limited either by the range of chemical and biological species that each can deliver, or by the efficiency or throughput of the delivery. While there have been previous efforts to use nanostructured materials to introduce bioactive species into cells, they too have been inefficient and generally limited to the delivery of genetic material. The potential to deliver a wide range of molecules at sites of one’s choosing lends itself to a broad variety of implementations and applications, including the following:
1. Massive, parallel screening for drug discovery: the cellular effects of many proteins (proteomics), siRNAs (knockdown efficacy), and small molecules (drug/antibiotic resistance) may be assayed simultaneously, either singly or in combinations; by perturbing different elements of a particular cellular pathway, the causal relationships between those elements can be discovered.
2. Cell-based ADME/Tox assays: the concentration-dependent effects of a particular protein, drug, and/or combinations can be studied in cell-based assays.
3. Derivation of induced pluripotent stem (iPS) cells: the platform is suitable for efficiently delivering (and discovering) molecules capable of reprogramming adult cells into iPS cells.
4. Designer cell networks and disease models: the platform can be used to construct complex cellular systems, such as synaptic pairs of differentially perturbed neurons for modeling neurodegenerative diseases.
5. Assays of epigenetic factors: the platform could be used to discover molecules that usefully affect the differentiation and development of any set of cells (e.g., stem cells, iPS cells, or differentiated tissues), and then further used to generate cells of a particular lineage for use in cell-based therapies.
Intellectual Property Status: Patent(s) Pending