Robert K. Delong

Functionalized gold nanoparticles for the binding, stabilization, and delivery of therapeutic DNA, RNA, and other biological macromolecules.

Nanotechnology has virtually exploded in the last few years with seemingly limitless opportunity across all segments of our society. If gene and RNA therapy are to ever realize their full potential, there is a great need for nanomaterials that can bind, stabilize, and deliver these macromolecular nucleic acids into human cells and tissues. Many researchers have turned to gold nanomaterials, as gold is thought to be relatively well tolerated in humans and provides an inert material upon which nucleic acids can attach. Here, we review the various strategies for associating macromolecular nucleic acids to the surface of gold nanoparticles (GNPs), the characterization chemistries involved, and the potential advantages of GNPs in terms of stabilization and delivery.

Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells

We have developed a method by which remarkably higher efficiencies of transient and stable transformation were achieved in bombardment transformation of plants. Over fivefold increase in transient gus gene expression was achieved when rice or maize suspension cells were bombarded with gold particles coated with plasmid DNA in the presence of protamine instead of the conventional spermidine. A 3.3-fold improvement in stable transformation efficiency was also observed using rice suspension cells with the new coating approach. The coated protamine-plasmid DNA complex resisted degradation by a DNase or by rice cell extract much longer than the spermidine-plasmid DNA complex. The results from this study suggest that protamine protects plasmid DNA longer than spermidine when being delivered inside the cells, probably by forming a nano-scale complex, and thus helps improve the efficiency of particle bombardment-mediated plant transformation.

Towards biosensors based on conducting polymer nanowires

AbstractWe report the electrochemical deposition of poly(pyrrolepropylic acid) nanowires, their covalent modification with antibodies and their conversion into potential functional sensor devices. The nanowires and the devices were characterised by optical microscopy, fluorescence microscopy, electron microscopy and electrical measurements. Fluorescence images, current–voltage (I–V) profiles and real-time sensing measurements demonstrated a rapid and highly sensitive and selective detection of human serum albumin (HSA), a substance that has been used to diagnose incipient renal disease. The detection is based on the selective binding of HSA onto anti-HSA that is covalently attached to the nanowires. The binding changes the electrical properties of the nanowires thus enabling the real-time detection. Whilst the utility of the research was demonstrated for protein binding/detection, the technology could easily be designed for the detection of other analytes by the modification of polymer nanowires with other analyte-specific molecules/biomolecules. Therefore, the technology has the potential to positively impact broad analytical applications in the biomedical, environmental and other sectors. FigureReal-time dynamic current response on sequential exposure of buffer, bovine serum albumin (BSA) and human serum albumin (HSA) onto anti-HSA modified poly (pyrrolepropylic acid) nanowires. Fluorescence images of poly(pyrrolepropylic acid) nanowire (top right) and polypyrrole nanowire control (bottom right) after sequential treatment with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), anti HSA and fluorophore-labeled HSA.

Characterization of biomolecular nanoconjugates by high-throughput delivery and spectroscopic difference

AIM Nanoparticle conjugates have the potential for delivering siRNA, splice-shifting oligomers or nucleic acid vaccines, and can be applicable to anticancer therapeutics. This article compares tripartite conjugates with gold nanoparticles or synthetic methoxypoly(ethylene glycol)-block-polyamidoamine dendrimers. MATERIALS & METHODS Interactions with model liposomes of a 1:1 molar ratio of tripalmitin:cholesterol or phospholipid:cholesterol were investigated by high-throughput absorbance, as well as fluorescence difference and cellular luminescence assays. RESULTS Spectral differences and dynamic light-scattering spectroscopy shifts demonstrated the interaction of conjugates with liposomes. Biological activity was demonstrated by upregulation of gene expression via splice-shifting oligomers, delivery of anti-B-Raf siRNA in cultured human cancer cells or tuberculosis antigen 85B plasmid expression vector in a coculture model of antigen presentation. CONCLUSION The data suggests that gold nanoparticles and methoxypoly(ethylene glycol)-block-polyamidoamine dendrimer nanoconjugates may have potential for binding, stabilization and delivery of splice-shifting oligomers, siRNA and nucleic acid vaccines for preclinical trials.

Characterization and performance of nucleic acid nanoparticles combined with protamine and gold.

Macromolecular nucleic acids such as DNA vaccines, siRNA, and splice-site switching oligomers (SSO) have vast chemotherapeutic potential. Nanoparticulate biomaterials hold promise for DNA and RNA delivery when a means for binding is identified that retains structure-function and provides stabilization by the nanoparticles. In order to provide these benefits of binding, we combined DNA and RNA with protamine-demonstrating association to gold microparticles by electrophoretic, gel shot, fluorescence, and dynamic laser light spectroscopy (DLLS). A pivotal finding in these studies is that the Au-protamine-DNA conjugates greatly stabilize the DNA; and DNA structure and vaccine activity are maintained even after exposure to physical, chemical, and temperature-accelerated degradation. Specifically, protamine formed nanoparticles when complexed to RNA. These complexes could be detected by gel shift and were probed by high throughput absorbance difference spectroscopy (HTADS). Biological activity of these RNA nanoparticles (RNPs) was demonstrated also by a human tumor cell splice-site switching assay and by siRNA delivery against B-Raf-a key cancer target. Finally, RNA:protamine particles inhibited growth of cultured human tumor cells and bacteria. These data provide new insights into DNA and RNA nanoparticles and prospects for their delivery and chemotherapeutic activity.

mPEG-PAMAM-G4 Nucleic Acid Nanocomplexes: Enhanced Stability, RNase Protection, and Activity of Splice Switching Oligomer and Poly I:C RNA

Dendrimer chemistries have virtually exploded in recent years with increasing interest in this class of polymers as gene delivery vehicles. An effective nucleic acid delivery vehicle must efficiently bind its cargo and form physically stable complexes. Most importantly, the nucleic acid must be protected in biological fluids and tissues, as RNA is extremely susceptible to nuclease degradation. Here, we characterized the association of nucleic acids with generation 4 PEGylated poly(amidoamine) dendrimer (mPEG-PAMAM-G4). We investigated the formation, size, and stability over time of the nanoplexes at various N/P ratios by gel shift and dynamic light scatter spectroscopy (DLS). Further characterization of the mPEG-PAMAM-G4/nucleic acid association was provided by atomic force microscopy (AFM) and by circular dichroism (CD). Importantly, mPEG-PAMAM-G4 complexation protected RNA from treatment with RNase A, degradation in serum, and various tissue homogenates. mPEG-PAMAM-G4 complexation also significantly enhanced the functional delivery of RNA in a novel engineered human melanoma cell line with splice-switching oligonucleotides (SSOs) targeting a recombinant luciferase transcript. mPEG-PAMAM-G4 triconjugates formed between gold nanoparticle (GNP) and particularly manganese oxide (MnO) nanorods, poly IC, an anticancer RNA, showed enhanced cancer-killing activity by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay.

Association of Poly I:C RNA and Plasmid DNA onto MnO Nanorods Mediated by PAMAM

In this study, manganese oxide (MnO) nanorods and its association with polyamidoamine dendrimer (PAMAM) and macromolecular RNA were analyzed. Because manganese is found naturally in cells and tissues and binds proteins and nucleic acids, nanomaterials derived from manganese, such as first generation MnO, may have potential as a biocompatible delivery agent for therapeutic or diagnostic biomedical applications. Nucleic acids have a powerful influence over cell processes, such as gene transcription and RNA processing; however, macromolecular RNA is particularly difficult to stabilize as a nanoparticle and to transport across cell membranes while maintaining structure and function. PAMAM is a cationic, branching dendrimer known to form strong complexes with nucleic acids and to protect them from degradation and is also considered to be a cell penetrating material. There is currently much interest in polyinosinic:polycytidylic RNA (poly I:C) because of its potent and specific immunogenic properties and as a solo or combination therapy. In order to address this potential, here, as a first step, we used PAMAM to attach poly I:C onto MnO nanorods. Morphology of the MnO nanorods was examined by field emission scanning electron microscopy (FESEM) and their composition by energy dispersive X-ray microanalysis (EDX). Evidence was generated for RNA:PAMAM:MnO nanorod binding by a gel shift assay using gel electrophoresis, a sedimentation assay using UV spectroscopy, and zeta potential shifts using dynamic laser light scattering. The data suggest that RNA was successfully attached to the MnO nanorods using PAMAM, and this suggestion was supported by direct visualization of the ternary complexes with FESEM characterizations. In order to confirm that the associations were biocompatible and taken up by cells, MTT assays were carried out to assess the metabolic activity of HeLa cells after incubation with the complexes and appropriate controls. Subsequently, we performed transfection assays using PAMAM:MnO complexes with pDNA encoding a green fluorescent protein reporter gene instead of RNA. The results suggest that the complexes had minimal impact on metabolic activity and were readily taken up by cells, and the fluorescent protein was expressed. From the evidence, we conclude that complexes of PAMAM:MnO interact with nucleic acids to form associations that are well-tolerated and readily taken up by cells.

Zinc Oxide Nanoparticle–Poly I:C RNA Complexes: Implication as Therapeutics against Experimental Melanoma

There is current interest in harnessing the combined anticancer and immunological effect of nanoparticles (NPs) and RNA. Here, we evaluate the bioactivity of poly I:C (pIC) RNA, bound to anticancer zinc oxide NP (ZnO-NP) against melanoma. Direct RNA association to unfunctionalized ZnO-NP is shown by observing change in size, zeta potential, and absorption/fluorescence spectra upon complexation. RNA corona was visualized by transmission electron microscopy (TEM) for the first time. Binding constant (Kb = 1.6-2.8 g-1 L) was determined by modified Stern-Volmer, absorption, and biological surface activity index analysis. The pIC-ZnO-NP complex increased cell death for both human (A375) and mouse (B16F10) cell lines and suppressed tumor cell growth in BALB/C-B16F10 mouse melanoma model. Ex vivo tumor analysis indicated significant molecular activity such as changes in the level of phosphoproteins JNK, Akt, and inflammation markers IL-6 and IFN-γ. High throughput proteomics analysis revealed zinc oxide and poly I:C-specific and combinational patterns that suggested possible utility as an anticancer and immunotherapeutic strategy against melanoma.

Two-Dimensional Fluorescence Difference Spectroscopy to Characterize Nanoparticles and their Interactions.

Two dimensional fluorescence difference spectroscopy (2D FDS) detects nanoparticle interactions following surface functionalization and biomolecule loading by generating a spectral signature of the fluorescent intensity per excitation and emission wavelengths. Comparing metal oxide nanoparticles revealed a unique spectral signature per material composition. 2D FDS showed to be sensitive to changes in surface properties between ZnO NPs synthesized by different methods. ZnO NP loaded with glycol chitosan, polyacrylic acid (PAA), or methoxy polyethylene glycol (mPEG) exhibited a distinct spectral signature shift. ZnO NP loaded with Torula Yeast RNA (TYRNA)(640 nm), polyinosinic: polycytidylic acid (pIC)(680 nm), or splice switching oligonucleotide (SSO)(650 nm) each revealed a shift in emission. Ras-Binding domain (RBD) at three concentrations (25, 37.5, 50 μg/mL) showed that fluorescent intensity was inversely related to the concentration of protein loaded. These data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions as a quality assurance tool.

Assembly of a Dual Aptamer Gold Nanoparticle Conjugate Ensemble inthe Specific Detection of Thrombin when Coupled with Dynamic LightScattering Spectroscopy

We demonstrate an extremely facile, rapid, specific and selective method for detecting proteins using aptamerconjugated gold nanoparticles coupled with dynamic light scattering (DLS) at ambient conditions. Addition of proteins to aptamer-conjugated gold nanoparticles (GNPs) induced the formation of protein-aptamer-gold nanoparticle conjugate complexes. The average hydrodynamic diameter of the nanoconjugate complexes as measured by DLS, increased with the corresponding increase in protein concentration. This correlation formed the analytical basis of the assay. The nanoparticles and nanoconjugate complexes were characterized by transmission electron microscopy, ultra-violet visible spectroscopy and DLS. Various parameters affecting the assay were optimized. Using thrombin as the model analyte, we demonstrated the detection of as low as 1.41 nM (0.05 μg/mL) of the protein. A linear dynamic range of up to 300 nM (11 μg/mL) was realized. The presence of other interfering proteins such as BSA showed no effect on the assay response.The presence of other interfering proteins such as bovine serum albumin (BSA) showed no significant effect on the assay response.