We describe the use of gold nanoparticle-oligonucleotide complexes as intracellular gene regulation agents for the control of protein expression in cells. These oligonucleotide-modified nanoparticles have affinity constants for complementary nucleic acids that are higher than their unmodified oligonucleotide counterparts, are less susceptible to degradation by nuclease activity, exhibit greater than 99% cellular uptake, can introduce oligonucleotides at a higher effective concentration than conventional transfection agents, and are nontoxic to the cells under the conditions studied. By chemically tailoring the density of DNA bound to the surface of gold nanoparticles, we demonstrated a tunable gene knockdown.

It was first shown more than ten years ago that DNA oligonucleotides can be attached to gold nanoparticles rationally to direct the formation of larger assemblies. Since then, oligonucleotide-functionalized nanoparticles have been developed into powerful diagnostic tools for nucleic acids and proteins, and into intracellular probes and gene regulators. In contrast, the conceptually simple yet powerful idea that functionalized nanoparticles might serve as basic building blocks that can be rationally assembled through programmable base-pairing interactions into highly ordered macroscopic materials remains poorly developed. So far, the approach has mainly resulted in polymerization, with modest control over the placement of, the periodicity in, and the distance between particles within the assembled material. That is, most of the materials obtained thus far are best classified as amorphous polymers, although a few examples of colloidal crystal formation exist. Here, we demonstrate that DNA can be used to control the crystallization of nanoparticle-oligonucleotide conjugates to the extent that different DNA sequences guide the assembly of the same type of inorganic nanoparticle into different crystalline states. We show that the choice of DNA sequences attached to the nanoparticle building blocks, the DNA linking molecules and the absence or presence of a non-bonding single-base flexor can be adjusted so that gold nanoparticles assemble into micrometre-sized face-centred-cubic or body-centred-cubic crystal structures. Our findings thus clearly demonstrate that synthetically programmable colloidal crystallization is possible, and that a single-component system can be directed to form different structures.

We have investigated the variables that influence DNA coverage on gold nanoparticles. The effects of salt concentration, spacer composition, nanoparticle size, and degree of sonication have been evaluated. Maximum loading was obtained by salt aging the nanoparticles to ~0.7 M NaCl in the presence of DNA containing a poly(ethylene glycol) (PEG) spacer. In addition, DNA loading was substantially increased by sonicating the nanoparticles during the surface loading process. Lastly, nanoparticles up to 250 nm in diameter were found have ~2 orders of magnitude higher DNA loading than smaller (13-30 nm) nanoparticles, a consequence of their larger surface area. Stable large particles are attractive for a variety of biodiagnostic assays.

Nanoparticles are employed for delivering therapeutics into cells1,2. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell specific internalization, excretion, toxicity, and efficacy3-7. A variety of materials have been explored for delivering small interfering RNAs (siRNAs) - a therapeutic agent that suppresses the expression of targeted genes8,9. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, lack of tissue specificity and potential toxicity10-12. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer targeting ligands (such as peptides and folate) on the nanoparticle surface can be precisely controlled. We show that at least three folate molecules per nanoparticle is required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t1/2 ∼ 24.2 min) than the parent siRNA (t1/2 ∼ 6 min).

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.drug delivery | induced pluripotent stem cells | reprogramming | protein delivery | nanoparticle delivery I ntracellular delivery of macromolecules is a critical step in therapeutic and research applications. Nanoparticle-mediated delivery of DNA and RNA, for example, is being explored for gene therapy (1, 2), while protein delivery is a promising means of affecting cellular function in both clinical (3) and laboratory (4) settings. Other materials, such as small molecules, quantum dots, or gold nanoparticles, are of interest for cancer therapies (5, 6), intracellular labeling (7,8), and single-molecule tracking (9).The cell membrane is largely impermeable to macromolecules. Many existing techniques use polymeric nanoparticles (10, 11), liposomes (12), or chemical modifications of the target molecule (13), such as cell-penetrating peptides (CPPs) (14, 15), to facilitate membrane poration or endocytotic delivery. In these methods, the delivery vehicle's efficacy is often dependent on the structure of the target molecule and the cell type. These methods are thus efficient in the delivery of structurally uniform materials, such as nucleic acids, but often ill-suited for the delivery of more structurally diverse materials, such as proteins (16,17) and some nanomaterials (7). Moreover, the endosome escape mechanism that most of these methods rely on is often inefficient; hence, much material remains trapped in endosomal and lysosomal vesicles (18). More effective gene delivery methods, such as viral vectors (19,20), however, often risk chromosomal integration and are limited to DNA and RNA delivery.Membrane poration methods, such as electroporation (21, 22) and sonoporation (23), are an attractive alternative in some applications. Indeed, electroporation has demonstrated its efficacy in a number of DNA (24) and ...