David A. Giljohann

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.

Oligonucleotide-Modified Gold Nanoparticles for Intracellular Gene Regulation

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.

Drivers of biodiagnostic development

The promise of point-of-care medical diagnostics — tests that can be carried out at the site of patient care — is enormous, bringing the benefits of fast and reliable testing and allowing rapid decisions on the course of treatment to be made. To this end, much innovation is occurring in technologies for use in biodiagnostic tests. Assays based on nanomaterials, for example, are now beginning to make the transition from the laboratory to the clinic. But the potential for such assays to become part of routine medical testing depends on many scientific factors, including sensitivity, selectivity and versatility, as well as technological, financial and policy factors.

Gene Regulation with Polyvalent siRNA-Nanoparticle Conjugates

We report the synthesis and characterization of polyvalent RNA-gold nanoparticle conjugates (RNA-Au NPs), nanoparticles that are densely functionalized with synthetic RNA oligonucleotides and designed to function in the RNAi pathway. The particles were rationally designed and synthesized to be free of degrading enzymes, have a high surface loading of siRNA duplexes, and contain an auxiliary passivating agent for increased stability in biological media. The resultant conjugates have a half-life six times longer than that of free dsRNA, readily enter cells without the use of transfection agents, and demonstrate a high gene knockdown capability in a cell model.

Polyvalent DNA Nanoparticle Conjugates Stabilize Nucleic Acids

Polyvalent oligonucleotide gold nanoparticle conjugates have unique fundamental properties including distance-dependent plasmon coupling, enhanced binding affinity, and the ability to enter cells and resist enzymatic degradation. Stability in the presence of enzymes is a key consideration for therapeutic uses; however the manner and mechanism by which such nanoparticles are able to resist enzymatic degradation is unknown. Here, we quantify the enhanced stability of polyvalent gold oligonucleotide nanoparticle conjugates with respect to enzyme-catalyzed hydrolysis of DNA and present evidence that the negatively charged surfaces of the nanoparticles and resultant high local salt concentrations are responsible for enhanced stability.

Aptamer nano-flares for molecular detection in living cells.

We demonstrate a composite nanomaterial, termed an aptamer nano-flare, that can directly quantify an intracellular analyte in a living cell. Aptamer nano-flares consist of a gold nanoparticle core functionalized with a dense monolayer of nucleic acid aptamers with a high affinity for adenosine triphosphate (ATP). The probes bind selectively to target molecules and release fluorescent reporters which indicate the presence of the analyte. Additionally, these nanoconjugates are readily taken up by cells where their signal intensity can be used to quantify intracellular analyte concentration. These nanoconjugates are a promising approach for the intracellular quantification of other small molecules or proteins, or as agents that use aptamer binding to elicit a biological response in living systems.

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.

Scavenger Receptors Mediate Cellular Uptake of Polyvalent Oligonucleotide-Functionalized Gold Nanoparticles

Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochemical analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.

Multiplexed nanoflares: mRNA detection in live cells.

We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.

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.