Soo-n Ryoo

Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies.

Carbon-based materials, including graphene and carbon nanotubes, have been considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for stem cell differentiation, and components of implant devices. Despite the potential biomedical applications of these materials, only limited information is available regarding the cellular events, including cell viability, adhesion, and spreading, that occur when mammalian cells interface with carbon-based nanomaterials. Here, we report behaviors of mammalian cells, specifically NIH-3T3 fibroblast cells, grown on supported thin films of graphene and carbon nanotubes to investigate biocompatibility of the artificial surface. Proliferation assay, cell shape analysis, focal adhesion study, and quantitative measurements of cell adhesion-related gene expression levels by RT-PCR reveal that the fibroblast cells grow well, with different numbers and sizes of focal adhesions, on graphene- and carbon nanotube-coated substrates. Interestingly, the gene transfection efficiency of cells grown on the substrates was improved up to 250% that of cells grown on a cover glass. The present study suggests that these nanomaterials hold high potential for bioapplications showing high biocompatibility, especially as surface coating materials for implants, without inducing notable deleterious effects while enhancing some cellular functions (i.e., gene transfection and expression).

Facile Synthesis of Monodispersed Mesoporous Silica Nanoparticles with Ultralarge Pores and Their Application in Gene Delivery

Among various nanoparticles, the silica nanoparticle (SiNP) is an attractive candidate as a gene delivery carrier due to advantages such as availability in porous forms for encapsulation of drugs and genes, large surface area to load biomacromolecules, biocompatibility, storage stability, and easy preparation in large quantity with low cost. Here, we report on a facile synthesis of monodispersed mesoporous silica nanoparticles (MMSN) possessing very large pores (>15 nm) and application of the nanoparticles to plasmid DNA delivery to human cells. The aminated MMSN with large pores provided a higher loading capacity for plasmids than those with small pores (∼2 nm), and the complex of MMSN with plasmid DNA readily entered into cells without supplementary polymers such as cationic dendrimers. Furthermore, MMSN with large pores could efficiently protect plasmids from nuclease-mediated degradation and showed much higher transfection efficiency of the plasmids encoding luciferase and green fluorescent protein (pLuc, pGFP) compared to MMSN with small pores (∼2 nm).

Quantitative and Multiplexed MicroRNA Sensing in Living Cells Based on Peptide Nucleic Acid and Nano Graphene Oxide (PANGO)

MicroRNA (miRNA) is an important small RNA which regulates diverse gene expression at the post-transcriptional level. miRNAs are considered as important biomarkers since abnormal expression of specific miRNAs is associated with many diseases including cancer and diabetes. Therefore, it is important to develop biosensors to quantitatively detect miRNA expression levels. Here, we develop a nanosized graphene oxide (NGO) based miRNA sensor, which allows quantitative monitoring of target miRNA expression levels in living cells. The strategy is based on tight binding of NGO with peptide nucleic acid (PNA) probes, resulting in fluorescence quenching of the dye that is conjugated to the PNA, and subsequent recovery of the fluorescence upon addition of target miRNA. PNA as a probe for miRNA sensing offers many advantages including high sequence specificity, high loading capacity on the NGO surface compared to DNA and resistance against nuclease-mediated degradation. The present miRNA sensor allowed the detection of specific target miRNAs with the detection limit as low as ~1 pM and the simultaneous monitoring of three different miRNAs in a living cell.

DYRK1A-mediated hyperphosphorylation of Tau. A functional link between Down syndrome and Alzheimer disease.

Most individuals with Down syndrome show early onset of Alzheimer disease (AD), resulting from the extra copy of chromosome 21. Located on this chromosome is a gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). One of the pathological hallmarks in AD is the presence of neurofibrillary tangles (NFTs), which are insoluble deposits that consist of abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the physiological significance of this phosphorylation, an analysis was made of the amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently generated. A significant increase in the amount of pT212 was found in the brains of DYRK1A transgenic mice when compared with age-matched littermate controls. We further examined whether Dyrk1A phosphorylates other Tau residues that are implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of Tau to promote microtubule assembly. Following this, using mammalian cells and DYRK1A TG mouse brains, it was demonstrated that the amounts of phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are high. These results provide the first in vivo evidence for a physiological role of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of the DYRK1A gene contributes to the early onset of AD.

Dual‐specificity tyrosine(Y)‐phosphorylation regulated kinase 1A‐mediated phosphorylation of amyloid precursor protein: evidence for a functional link between Down syndrome and Alzheimer’s disease

Most individuals with Down Syndrome (DS) show an early‐onset of Alzheimer’s disease (AD), which potentially results from the presence of an extra copy of a segment of chromosome 21. Located on chromosome 21 are the genes that encode β‐amyloid (Aβ) precursor protein (APP ), a key protein involved in the pathogenesis of AD, and dual‐specificity tyrosine(Y)‐phosphorylation regulated kinase 1A (DYRK1A ), a proline‐directed protein kinase that plays a critical role in neurodevelopment. Here, we describe a potential mechanism for the regulation of AD pathology in DS brains by DYRK1A‐mediated phosphorylation of APP. We show that APP is phosphorylated at Thr668 by DYRK1A in vitro and in mammalian cells. The amounts of phospho‐APP and Aβ are increased in the brains of transgenic mice that over‐express the human DYRK1A protein. Furthermore, we show that the amounts of phospho‐APP as well as those of APP and DYRK1A are elevated in human DS brains. Taken together, these results reveal a potential regulatory link between APP and DYRK1A in DS brains, and suggest that the over‐expression of DYRK1A in DS may play a role in accelerating AD pathogenesis through phosphorylation of APP.

Synergistic Effect of Graphene Oxide/MWCNT Films in Laser Desorption/Ionization Mass Spectrometry of Small Molecules and Tissue Imaging

Matrix-assisted laser desorption/ionization mass spectrometry has been considered an important tool for various biochemical analyses and proteomics research. Although addition of conventional matrix efficiently supports laser desorption/ionization of analytes with minimal fragmentation, it often results in high background interference and misinterpretation of the spatial distribution of biomolecules especially in low-mass regions. Here, we show design, systematic characterization, and application of graphene oxide/multiwalled carbon nanotube-based films fabricated on solid substrates as a new matrix-free laser desorption/ionization platform. We demonstrate that the graphene oxide/multiwalled carbon nanotube double layer provides many advantages as a laser desorption/ionization substrate, such as efficient desorption/ionization of analytes with minimum fragmentation, high salt tolerance, no sweet-spots for mass signal, excellent durability against mechanical and photoagitation and prolonged exposure to ambient conditions, and applicability to tissue imaging mass spectrometry. This platform will be widely used as an important tool for mass spectrometry-based biochemical analyses because of its outstanding performance, long-term stability, and cost effectiveness.

Efficient functional delivery of siRNA using mesoporous silica nanoparticles with ultralarge pores.

Among various nanoparticles, mesoporous silica nanoparticles (MSNs) have attracted extensive attention for developing efficient drug-delivery systems, mostly due to their high porosity and biocompatibility. However, due to the small pore size, generally below 5 nm in diameter, potential drugs that are loaded into the pore have been limited to small molecules. Herein, a small interfering RNA (siRNA) delivery strategy based on MSNs possessing pores with an average diameter of 23 nm is presented. The siRNA is regarded as a powerful gene therapeutic agent for treatment of a wide range of diseases by enabling post-transcriptional gene silencing, so-called RNA interference. Highly efficient, sequence-specific, and technically very simple target gene knockdown is demonstrated using MSNs with ultralarge pores of size 23 nm in vitro and in vivo without notable cytotoxicity.

The effective nuclear delivery of doxorubicin from dextran-coated gold nanoparticles larger than nuclear pores

To date, gold nanoparticles (AuNPs) have been investigated for diverse bioapplications. Generally, AuNPs are engineered to possess surface coating with organic/inorganic shells to increase colloidal stability in biological solutions and to facilitate chemical conjugation. In the present study, we developed a strategy to prepare dextran-coated AuNPs with control over its size by simply boiling an aqueous solution of Au salt and dextran, in which dextran serves as both reducing agent for AuNP (Au(0)) formation from Au(III) and AuNP surface coating material. The prepared dextran-coated AuNPs (dAuNPs) maintained its colloidal stability under high temperature, high salt concentration, and extreme pH. Importantly, the dAuNP remarkably improved efficacy of an anti-cancer agent, doxorubicin (Dox), when harnessed as a Dox delivery carrier. The half-maximal inhibitory concentration (EC50) of Dox-conjugated dAuNP with diameter of 170 nm was ∼9 pM in HeLa cells, which was 1.1 × 10(5) times lower than that of free Dox and lower than any previously reported values of Dox-nanoparticle complex. Interestingly, smaller AuNPs with 30 and 70 nm showed about 10 times higher EC50 than 170 nm AuNPs when treated to HeLa cells after conjugation with Dox. To achieve high cytotoxicity as cancer therapeutics, Dox should be delivered into nucleus to intercalate with DNA double helix. We show here that Dox-AuNPs was far more efficient as an anti-cancer drug than free Dox by releasing from AuNPs through spontaneous degradation of dextran, allowing free diffusion and nuclear uptake of Dox. We also revealed that larger AuNPs with lower degree of dextran crosslinking promoted faster degradation of dextran shells.

Discovery of Hepatitis C Virus NS3 Helicase Inhibitors by a Multiplexed, High‐Throughput Helicase Activity Assay Based on Graphene Oxide

A GO‐to solution: A simple graphene oxide (GO)‐based assay to screen for selective inhibitors of a hepatitis C virus (HCV) helicase along with inhibitors of a severe acute respiratory syndrome coronavirus (SARS CoV) helicase was tested (see scheme). A single screen found five inhibitors highly selective for the HCV helicase orthologous to the SARS CoV helicase. Some of these hits were validated using the same GO‐based assay.WILEY-VCH

Functional delivery of DNAzyme with iron oxide nanoparticles for hepatitis C virus gene knockdown

DNAzyme is an attractive therapeutic oligonucleotide which enables cleavage of mRNA in a sequence-specific manner and thus, silencing target gene. A particularly important challenge in achieving the successful down-regulation of gene expression is to efficiently deliver DNAzymes to disease sites and cells. Here, we report the nanoparticle-assisted functional delivery of therapeutic DNAzyme for the treatment of hepatitis C by inducing knockdown of hepatitis C virus (HCV) gene, NS3. HCV NS3 gene encodes helicase and protease which are essential for the virus replication. The nanocomplex showed efficient NS3 knockdown while not evoking undesired immune responses or notable cytotoxicity. We also demonstrated the DNAzyme conjugated nanoparticle system could be applicable in vivo by showing the accumulation of the nanoparticles in liver, and more specifically, in hepatocytes. We believe that the present work is a successful demonstration of effective, functional, non-immunostimulatory DNAzyme delivery system based on inorganic nanoparticles with high potential for further therapeutic application of DNAzyme in the treatment of hepatitis C.