Gold colloids have fascinated scientists for over a century and are now heavily utilized in chemistry, biology, engineering, and medicine. Today these materials can be synthesized reproducibly, modified with seemingly limitless chemical functional groups, and, in certain cases, characterized with atomic-level precision. This Review highlights recent advances in the synthesis, bioconjugation, and cellular uses of gold nanoconjugates. There are now many examples of highly sensitive and selective assays based upon gold nanoconjugates. In recent years, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to molecule-based systems, but rather for their new physical and chemical properties, which confer substantive advantages in cellular and medical applications.

Topical application of nucleic acids offers many potential therapeutic advantages for suppressing genes in the skin, and potentially for systemic gene delivery. However, the epidermal barrier typically precludes entry of gene-suppressing therapy unless the barrier is disrupted. We now show that spherical nucleic acid nanoparticle conjugates (SNA-NCs), gold cores surrounded by a dense shell of highly oriented, covalently immobilized siRNA, freely penetrate almost 100% of keratinocytes in vitro, mouse skin, and human epidermis within hours after application. Significantly, these structures can be delivered in a commercial moisturizer or phosphate-buffered saline, and do not require barrier disruption or transfection agents, such as liposomes, peptides, or viruses. SNANCs targeting epidermal growth factor receptor (EGFR), an important gene for epidermal homeostasis, are >100-fold more potent and suppress longer than siRNA delivered with commercial lipid agents in cultured keratinocytes. Topical delivery of 1.5 uM EGFR siRNA (50 nM SNA-NCs) for 3 wk to hairless mouse skin almost completely abolishes EGFR expression, suppresses downstream ERK phosphorylation, and reduces epidermal thickness by almost 40%. Similarly, EGFR mRNA in human skin equivalents is reduced by 52% after 60 h of treatment with 25 nM EGFR SNA-NCs. Treated skin shows no clinical or histological evidence of toxicity. No cytokine activation in mouse blood or tissue samples is observed, and after 3 wk of topical skin treatment, the SNA structures are virtually undetectable in internal organs. SNA conjugates may be promising agents for personalized, topically delivered gene therapy of cutaneous tumors, skin inflammation, and dominant negative genetic skin disorders.T he recent development of small molecule inhibitors and antibodies that target components of signaling pathways has revolutionized the treatment of cancers, inflammatory diseases, and genetic disorders, including those that largely manifest in skin (1-3). These protein-based therapeutics, however, are costly, have limited targeting ability, and, when delivered through traditional intravenous or gastrointestinal routes, can lead to systemic toxicity (4, 5). Topical delivery is particularly attractive for the therapy of skin disorders. However, proteins larger than a few hundred daltons cannot easily enter the skin, and high concentrations of proteins must be applied for a cutaneous effect (6). An alternative to protein-based pathway inhibition involves the blocking and/or degradation of precursor mRNA before translation into protein. Gene silencing leads to down-regulation of protein expression and functions with greater specificity than inhibitors of protein function (7). In fact, targeted gene suppression by antisense DNA and siRNA has shown promising preclinical results, and/or is currently in clinical trials for a variety of diseases, including many forms of cancer (e.g., melanoma, neuroblastoma, and pancreatic adenocarcinoma), genetic disorders, and macular degeneration (8). For gene su...

We build off the previously described concept of a nano-flare to develop an oligonucleotide gold nanoparticle conjugate that is capable of both detecting and regulating intracellular levels of mRNA. We characterize the binding rate and specificity of these materials using survivin, a gene associated with the diagnosis and treatment of cancer, as a target. The nanoconjugate enters cells and binds mRNA, thereby decreasing the relative abundance of mRNA in a dose-and sequence-dependent manner and resulting in a fluorescent response. This represents the first demonstration of a single material capable of both mRNA regulation and detection. Further, we investigate the intracellular biochemistry of the nanoconjugate, elucidating its mechanism of gene regulation. This work is important to the study of biologically active nanomaterials such as the nano-flare and is a first step towards the development of an mRNA responsive 'theranostic'. KeywordsNanoparticle; oligonucleotide; mRNA; detection; gene regulation; theranostic Over the past decade, researchers have investigated the conjugation of biomolecules to inorganic nanomaterials, which has led to the development of hybrid materials with new activities. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] One important class of hybrid nanomaterial is composed of a gold nanoparticle functionalized with a dense monolayer of oligonucleotides. These polyvalent nanoconjugates have many interesting properties, including distance-dependent optical features, 21,22 enhanced nucleic acid binding, 23 resistance to degradation, 24 and the ability to enter cells without use of transfection agents. 25 These remarkable properties have enabled controlled assembly of materials, 26-28 molecular diagnostics, [29][30][31][32] and intracellular studies. [33][34][35] Materials with both regulation and detection capabilities are of growing interest for use in personalized medicine. 36 These 'theranostic' materials have the potential to both treat and diagnose disease and are useful for investigating intracellular events (i.e. target recognition and control of biological function). Traditionally, antisense oligonucleotides and molecular beacons have been used to regulate and detect intracellular mRNA, respectively. Antisense oligonucleotides regulate gene expression by binding target mRNA and preventing translation. 37 Molecular beacons detect nucleic acids by coupling a binding event with a signal transduction mechanism, such as the separation of a fluorophore-quencher pair. 38 Given that cell entry and mRNA binding are the first steps in both processes, it should be possible to design a single material for both regulation and detection. To the best of our knowledge, however, a material capable of both mRNA regulation and detection has not been reported. 39 Such materials must be readily taken up by cells, stable in intracellular environments, capable of binding nucleic acids, and possess a switchable signal that can be conveniently detected. NIH Public Access RES...

We report the development of a powerful analytical method that utilizes a tilted elastomeric pyramidal pen array in the context of a scanning probe lithography experiment to rapidly prepare libraries having as many as 25 million features over large areas with a range of feature sizes from the nano-to microscale. This technique can be used to probe important chemical and biological processes, opening up the field of nanocombinatorics. In a proof-of-concept investigation of mesenchymal stem cell (MSC) differentiation, combinatorial patterns first enabled a rapid and systematic screening of MSC adhesion, as a function of feature size, while uniform patterns were used to study differentiation with statistically significant sample sizes. Without media containing osteogenic-inducing chemical cues, cells cultured on nanopatterned fibronectin substrates direct MSC differentiation towards osteogenic fates when compared to nonpatterned fibronectin substrates. This powerful and versatile approach enables studies of many systems spanning biology, chemistry, and engineering areas.nanopatterning | polymer pen lithography | focal adhesions | osteogenesis | stem cell differentiation I t is important to note and distinguish that while the prevailing emphasis in lithography concerns the exact replication of structures across large areas, methods to rapidly generate combinatorial libraries having a tunable gradient of feature sizes with a single, maskless tool would be powerful for systematic investigations in fields spanning biology, materials engineering, and nanotechnology (1). Herein we report the development of a powerful and unique analytical method that utilizes a tilted elastomeric pyramidal pen array in the context of a scanning probe lithography experiment to rapidly prepare libraries having as many as 25 million features over large areas with a range of feature sizes from the nanoto microscale that can be used to probe important chemical and biological processes, opening up the field of nanocombinatorics. In a proof-of-concept investigation of mesenchymal stem cell (MSC) differentiation, combinatorial patterns first enabled a rapid and systematic screening of MSC adhesion, as a function of feature size, while uniform patterns were used to study differentiation with statistically significant sample sizes. Without media containing osteogenic-inducing chemical cues, cells cultured on nanopatterned fibronectin substrates direct MSC differentiation towards osteogenic fates when compared to nonpatterned fibronectin substrates.Polymer pen lithography (PPL) is a massively parallel directwrite technique that uses an array of elastomeric pyramidal pens to deliver materials (e.g., alkanethiols, proteins) to a surface and generate customizable patterns with feature sizes ranging from the nano-to microscale simply by changing (i) the amount of force applied to the pen array or (ii) the tip-substrate contact time (2-5). Indeed, PPL has already been shown to be a high-throughput patterning technique capable of generating arbitrary des...

The immune response of macrophage cells to internalized polyvalent nucleic acid-functionalized gold nanoparticles has been studied. This study finds that the innate immune response (as measured by interferon-β levels) to densely functionalized, oligonucleotide-modified nanoparticles is significantly less (up to a 25-fold decrease) when compared to a lipoplex carrying the same DNA sequence. The magnitude of this effect is inversely proportional to oligonucleotide density. It is proposed that the enzymes involved in recognizing foreign nucleic acids and triggering the immune response are impeded due to the local surface environment of the particle, in particular high charge density. The net effect is an intracelluar gene regulation agent that elicits a significantly lower cellular immune response than conventional DNA transfection materials.