Dual bioluminescence and near-infrared fluorescence monitoring to evaluate spherical nucleic acid nanoconjugate activity in vivo.

RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood-brain barrier-/blood-tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.

Design Considerations for RNA Spherical Nucleic Acids (SNAs).

Ribonucleic acids (RNAs) are key components in many cellular processes such as cell division, differentiation, growth, aging, and death. RNA spherical nucleic acids (RNA-SNAs), which consist of dense shells of double-stranded RNA on nanoparticle surfaces, are powerful and promising therapeutic modalities because they confer advantages over linear RNA such as high cellular uptake and enhanced stability. Due to their three-dimensional shell of oligonucleotides, SNAs, in comparison to linear nucleic acids, interact with the biological environment in unique ways. Herein, the modularity of the RNA-SNA is used to systematically study structure-function relationships in order to understand how the oligonucleotide shell affects interactions with a specific type of biological environment, namely, one that contains serum nucleases. We use a combination of experiment and theory to determine the key architectural properties (i.e., sequence, density, spacer moiety, and backfill molecule) that affect how RNA-SNAs interact with serum nucleases. These data establish a set of design parameters for SNA architectures that are optimized in terms of stability.

Enzymatically Controlled Vacancies in Nanoparticle Crystals.

In atomic systems, the mixing of metals results in distinct phase behavior that depends on the identity and bonding characteristics of the atoms. In nanoscale systems, the use of oligonucleotides as programmable "bonds" that link nanoparticle "atoms" into superlattices allows for the decoupling of atom identity and bonding. While much research in atomic systems is dedicated to understanding different phase behavior of mixed metals, it is not well understood on the nanoscale how changes in the nanoscale "bond" affect the phase behavior of nanoparticle crystals. In this work, the identity of the atom is kept the same, but the chemical nature of the bond is altered, which is not possible in atomic systems, through the use of DNA and RNA bonding elements. These building blocks assemble into single crystal nanoparticle superlattices with mixed DNA and RNA bonding elements throughout. The nanoparticle crystals can be dynamically changed through the selective and enzymatic hydrolysis of the RNA bonding elements, resulting in superlattices that retain their crystalline structure and habit, while incorporating up to 35% random vacancies generated from the nanoparticles removed. Therefore, the bonding elements of nanoparticle crystals can be enzymatically and selectively addressed without affecting the nature of the atom.

Modular and Chemically Responsive Oligonucleotide "Bonds" in Nanoparticle Superlattices.

Chemical bonds are a key determinant of the structure and properties of a material. Thus, rationally designing arbitrary materials requires complete control over the bond. While atomic bonding is dictated by the identity of the atoms, nanoparticle superlattice engineering, where nanoparticle "atoms" are held together by DNA "bonds", offers a route to design crystal lattices in a way that nature cannot: through altering the oligonucleotide bond. Herein, the use of RNA, as opposed to DNA, is explored by synthesizing superlattices in which nanoparticles are bonded by DNA/DNA, RNA/RNA, and DNA/RNA duplexes. By moving beyond nanoparticle superlattices assembled only with DNA, a new degree of freedom is introduced, providing programmed responsiveness to enzymes and greater bond versatility. Therefore, the oligonucleotide bond can have programmable function beyond dictating the structure of the material and moves nanoparticle superlattices closer to naturally occurring biomaterials, where the line between structural and functional elements is blurred.

Therapeutic applications of spherical nucleic acids.

Spherical nucleic acids (SNAs) represent an emerging class of nanoparticle-based therapeutics. SNAs consist of densely functionalized and highly oriented oligonucleotides on the surface of a nanoparticle which can either be inorganic (such as gold or platinum) or hollow (such as liposomal or silica-based). The spherical architecture of the oligonucleotide shell confers unique advantages over traditional nucleic acid delivery methods, including entry into nearly all cells independent of transfection agents and resistance to nuclease degradation. Furthermore, SNAs can penetrate biological barriers, including the blood-brain and blood-tumor barriers as well as the epidermis, and have demonstrated efficacy in several murine disease models in the absence of significant adverse side effects. In this chapter, we will focus on the applications of SNAs in cancer therapy as well as discuss multimodal SNAs for drug delivery and imaging.

Probing the inherent stability of siRNA immobilized on nanoparticle constructs.

Small interfering RNA (siRNA) is a powerful and highly effective method to regulate gene expression in vitro and in vivo. However, the susceptibility to serum nuclease-catalyzed degradation is a major challenge and it remains unclear whether the strategies developed to improve the stability of siRNA free in serum solution are ideal for siRNA conjugated to nanoparticle surfaces. Herein, we use spherical nucleic acid nanoparticle conjugates, consisting of gold nanoparticles (AuNPs) with siRNA chemisorbed to the surface, as a platform to study how a model siRNA targeting androgen receptor degrades in serum (SNA-siRNAAR). In solutions of 10% (vol/vol) FBS, we find rapid endonuclease hydrolysis at specific sites near the AuNP-facing terminus of siRNAAR, which were different from those of siRNAAR free in solution. These data indicate that the chemical environment of siRNA on a nanoparticle surface can alter the recognition of siRNA by serum nucleases and change the inherent stability of the nucleic acid. Finally, we demonstrate that incorporation of 2'-O-methyl RNA nucleotides at sites of nuclease hydrolysis on SNA-siRNAAR results in a 10-fold increase in siRNA lifetime. These data suggest that strategies for enhancing the serum stability of siRNA immobilized to nanoparticles must be developed from a dedicated analysis of the siRNA-nanoparticle conjugate, rather than a reliance on strategies developed for siRNA free in solution. We believe these findings are important for fundamentally understanding interactions between biological media and oligonucleotides conjugated to nanoparticles for the development of gene regulatory and therapeutic agents in a variety of disease models.

Self-assembling peptide assemblies bound to ZnS nanoparticles and their interactions with mammalian cells.

Self-assembling peptide sequences (both synthetic and natural) have emerged as a new group of building blocks for diverse applications. In this work we investigated the formation of assemblies of three diverse peptide sequences derived from the crustacean cardioactive peptide CCAP (1-9), a cardioaccelerator and neuropeptide transmitter in crustaceans, atrial natriuretic hormone ANP (1-28), a powerful vasodilator secreted by heart muscle cells of mammals, as well as adamstsostatin peptide ADS (1-17), which functions as an inhibitor of angiogenesis. The formation of assemblies was found to be dependent upon the sequences as well as the pH in which the assemblies were grown. The secondary structural transformation of the peptides was studied by circular dichroism as well as FTIR spectroscopy. In order to render the sequences luminescent, we conjugated the assemblies with ZnS nanoparticles. Finally the interactions of the peptide bound ZnS nanoparticles with mammalian normal rat kidney cells were explored. In some cases the nanoconjugates were found to adhere not only to the cellular membranes but also extend into the cytoplasm. Thus, such nanocomposites may be utilized for cell penetration and have the potential to serve as coercive multifunctional vectors for bioimaging and cellular delivery.

Biomimetic formation of chicoric-acid-directed luminescent silver nanodendrites.

In this work, we report the formation of well-defined silver nanodendrites via biomineralization under mild conditions in a single step, in the presence of the plant phytohormone chicoric acid (CA), a well-known HIV-I integrase inhibitor. CA played a dual role as reductant as well as directed the growth of the nanodendrites, which were found to grow primarily in the [111] and [200] directions. In addition to the formation of highly ordered hierarchical structures, the formed Ag nanodendrites were found to exhibit luminescence, as observed by confocal microscopy. This study not only demonstrates a new method for the preparation of luminescent silver nanodendrites using a simple, environmentally friendly biological method, but also indicates the ability of CA, a potent HIV-integrase inhibitor, to interact with silver ions which may shed light on its potential for additional biomedical and biosensor applications.

Fabrication of ellagic acid incorporated self-assembled peptide microtubes and their applications.

Ellagic acid (EA), a plant polyphenol known for its wide-range of health benefits was encapsulated within self-assembled threonine based peptide microtubes. The microtubes were assembled using the synthesized precursor bolaamphiphile bis(N-α-amido threonine)-1,5-pentane dicarboxylate. The self-assembly of the microstructures was probed at varying pH. In general, tubular formations were observed at a pH range of 4-6. The formed microtubes were then utilized for fabrication with EA. We probed the ability of the microtubes as drug release vehicles for EA as well as for antibacterial applications. It was found that the release of EA was both pH and concentration dependent. The biocompatibility as well as cytotoxicity of the EA-fabricated microtubes was examined in the presence of mammalian normal rat kidney (NRK) cells. Finally the antibacterial effects of the EA incorporated peptide microtubes was examined against Escherichia coli and Staphylococcus aureus.

Fabrication of collagen-elastin-bound peptide microtubes for mammalian cell attachment.

In this work we have designed self-assembled peptide-based microconstructs and examined their interactions with elastin and collagen for potential application as scaffolds for chondrocyte cell attachment. Being biological in nature, peptide-based nano- and microstructures have intrinsic molecular recognition properties which allow extensive chemical, conformational and functional diversity. We have synthesized a new peptide bolaamphiphile, bis(N-α-amido-val)-1,5-pentane dicarboxylate, and examined its self-assembly at varying pH values. The formation of high-density networks of nano- and microtubular structures was found to be in the range of pH 4-6. The formed microtubes were then covalently bound to varying concentrations of the extracellular matrix protein elastin, a versatile protein that allows for an extensive array of physical and chemical modifications to attune properties towards diverse necessities of biomedical applications. We found that binding to microtubes was concentration dependent. The morphological and chemical changes complementing the processes of self-assembly and binding to elastin were examined by electron microscopic and spectroscopic methods. Furthermore, we also incorporated the extracellular matrix protein type-I collagen, a critical constituent for designing biocompatible scaffolds, into the elastin functionalized micro-tubes. Since the main goal is to develop highly biocompatible protein functionalized microstructures that support cellular interactions, we examined the interactions of the microcomposites with chondrocyte cell line, in order to assess the biocompatibility and interaction between the microconstructs and the cells. The designed elastin and collagen-bound peptide microtubes may potentially serve as a new class of biomaterials by promoting cell growth and proliferation.

pH dependent spontaneous growth of ellagic acid assemblies for targeting HeLa cells.

Herein, we have studied the self-assembly and the spontaneous growth of microassemblies of the plant polyphenol ellagic acid for HeLa cancer cell imaging and therapy. The growth of the assemblies was studied at varying pH over time. It was found that initially microspheres were formed which gradually transformed into microfibers via nucleation and polymerization process. The optimum growth of microfibers was found to be in the pH range of 6-8. We have shown that the microfibers successfully adhered to the HeLa cell membranes and inhibited their proliferation. This biological approach, using assemblies derived from plant polyphenols, may be used for direct cellular drug delivery and may potentially help develop a simple and economical method to create building blocks with desired properties for a new generation of sensors, bioimaging and drug delivery systems.

Ellagic acid promoted biomimetic synthesis of shape-controlled silver nanochains.

In this work, ellagic acid (EA), a naturally occurring plant polyphenol, was utilized for the biomimetic synthesis of silver (Ag) nanoparticles, which over a period of time formed extended branched nanochains of hexagonal-shaped silver nanoparticles. It was found that EA not only has the capability of reducing silver ions, resulting in the formation of Ag nanoparticles, due to its extended polyphenolic system, but also appears to recognize and affect the Ag nanocrystal growth on the (111) face, leading to the formation of hexagon-shaped Ag nanocrystals. Initially, various Ag nanocrystal shapes were observed; however, over a longer period of time, a majority of hexagonal-shaped nanocrystals were formed. Although the exact mechanism of formation of the nanocrystals is not known, it appears that EA attaches to the silver nuclei, leading to lower surface energy of the (111) face. Further, the nanocrystals fuse together, forming interfaces among the aggregates, and, with time, those interfaces become lesser, and the nanoparticles merge together and share the same single crystallographic orientation, which leads to the formation of long elongated chains of hexagonal nanoparticles. This biomimetic approach may be developed as a green synthetic method to prepare building blocks with tunable properties for the development of nanodevices. Further, we explored the antibacterial properties and found that the tandem of EA-Ag nanochains substantially enhanced the antibacterial properties of both gram-positive and gram-negative bacteria compared to silver nanoparticles or EA alone. Additionally, the materials were also utilized for imaging of mammalian NRK (normal rat kidney) cells.