Pinal C. Patel

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.

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.

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.

Nanoparticle Shape Anisotropy Dictates the Collective Behavior of Surface-Bound Ligands

We report on the modification of the properties of surface-confined ligands in nanoparticle systems through the introduction of shape anisotropy. Specifically, triangular gold nanoprisms, densely functionalized with oligonucleotide ligands, hybridize to complementary particles with an affinity that is several million times higher than that of spherical nanoparticle conjugates functionalized with the same amount of DNA. In addition, they exhibit association rates that are 2 orders of magnitude greater than those of their spherical counterparts. This phenomenon stems from the ability of the flat, extended facets of nonspherical nanoparticles to (1) support more numerous ligand interactions through greater surface contact with complementary particles, (2) increase the effective local concentration of terminal DNA nucleotides that mediate hybridization, and (3) relieve the conformational stresses imposed on nanoparticle-bound ligands participating in interactions between curved surfaces. Finally, these same trends are observed for the pH-mediated association of nanoparticles functionalized with carboxylate ligands, demonstrating the generality of these findings.

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.

Peptide antisense nanoparticles

We have designed a heterofunctionalized nanoparticle conjugate consisting of a 13-nm gold nanoparticle (Au NP) containing both antisense oligonucleotides and synthetic peptides. The synthesis of this conjugate is accomplished by mixing thiolated oligonucleotides and cysteine-terminated peptides with gold nanoparticles in the presence of salt, which screens interactions between biomolecules, yielding a densely functionalized nanomaterial. By controlling the stoichiometry of the components in solution, we can control the surface loading of each biomolecule. The conjugates are prepared easily and show perinuclear localization and an enhanced gene regulation activity when tested in a cellular model. This heterofunctionalized structure represents a new strategy for preparing nanomaterials with potential therapeutic applications.

Nucleic acid-gold nanoparticle conjugates as mimics of microRNA.

Novel conjugates of gold nanoparticles (13±1nm) functionalized with synthetic microRNAs can enter cells without the aid of cationic co-carriers and mimic the function of endogenous microRNAs. These conjugates can regulate multiple proteins through interactions with 3′ untranslated region of the target mRNA and control cell behavior. The conjugates are a promising new tool for studying miRNA function and new candidates for miRNA replacement therapies.

Exosome encased spherical nucleic acid gold nanoparticle conjugates as potent microRNA regulation agents.

Exosomes are a class of naturally occurring nanomaterials that play crucial roles in the protection and transport of endogenous macromolecules, such as microRNA and mRNA, over long distances. Intense effort is underway to exploit the use of exosomes to deliver synthetic therapeutics. Herein, transmission electron microscopy is used to show that when spherical nucleic acid (SNA) constructs are endocytosed into PC-3 prostate cancer cells, a small fraction of them (<1%) can be naturally sorted into exosomes. The exosome-encased SNAs are secreted into the extracellular environment from which they can be isolated and selectively re-introduced into the cell type from which they were derived. In the context of anti-miR21 experiments, the exosome-encased SNAs knockdown miR-21 target by approximately 50%. Similar knockdown of miR-21 by free SNAs requires a ≈3000-fold higher concentration.