Scanning probe-enabled nanocombinatorics define the relationship between fibronectin feature size and stem cell fate.

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.

Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation.

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. SNA-NCs 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.

Gold nanoparticles for biology and medicine.

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.

Regulating immune response using polyvalent nucleic acid-gold nanoparticle conjugates.

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-beta 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.

Cellular response of polyvalent oligonucleotide-gold nanoparticle conjugates.

Nanoparticles are finding utility in myriad biotechnological applications, including gene regulation, intracellular imaging, and medical diagnostics. Thus, evaluating the biocompatibility of these nanomaterials is imperative. Here we use genome-wide expression profiling to study the biological response of HeLa cells to gold nanoparticles functionalized with nucleic acids. Our study finds that the biological response to gold nanoparticles stabilized by weakly bound surface ligands is significant (cells recognize and react to the presence of the particles), yet when these same nanoparticles are stably functionalized with covalently attached nucleic acids, the cell shows no measurable response. This finding is important for researchers studying and using nanomaterials in biological settings, as it demonstrates how slight changes in surface chemistry and particle stability can lead to significant differences in cellular responses.

Nano-flares for mRNA regulation and detection.

We build off the previously described concept of a nanoflare 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, 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 nanoflare and is a first step toward the development of an mRNA responsive "theranostic".