Taegyeong Kang

The effects of sub-lethal concentrations of silver nanoparticles on inflammatory and stress genes in human macrophages using cDNA microarray analysis.

Because of the limited information on size-dependent particle-mediated effects, the present study was conducted to determine if the changes in induced protein expression between 5 nm silver nanoparticles and 100 nm particles after exposure to sub-lethal concentrations. A total of 28,000 cDNA profiles were screened using 5 nm silver nanoparticles and 100 nm silver nanoparticles in a macrophage cell line. Based on results obtained from cDNA microarray we also assessed protein levels of hemeoxygenase-1 (HO-1), heat shock protein-70 (HSP-70) and interleukin-8 (IL-8), which were shown to significantly increase. Together with results obtained using N-acetylcystein (NAC), we were able to clearly show that low level and early stage exposure to 5 nm silver nanoparticles, but not 100 nm, induces expression of IL-8 as well as stress genes against reactive oxygen species (ROS). Therefore, we provide important data to understand and identify the early effects of silver nanoparticles on the immune system.

Vascular tube formation and angiogenesis induced by polyvinylpyrrolidone-coated silver nanoparticles.

Silver nanoparticles (AgNPs) are one of the most commonly used nanomaterials due to their antibacterial properties. In this study, we examined the effects of polyvinylpyrrolidone (PVP)-coated AgNPs (average size 2.3nm) on angiogenesis in both an in vivo model and an in vitro endothelial cell line, SVEC4-10. Increased angiogenesis was detected around the injection site of AgNP-containing Matrigel in vivo. AgNPs also increased the infiltration of endothelial cells and the hemoglobin (Hb) content in AgNP-Matrigel plugs implanted into mice. AgNPs induced endothelial cell tube formation on growth factor-reduced Matrigel, production of reactive oxygen species (ROS), and production of angiogenic factors, such as vascular endothelial growth factor (VEGF) and nitric oxide (NO), in SVEC4-10 cells. In addition, AgNPs promoted the activation of FAK, Akt, ERK1/2, and p38, which are all involved in VEGF receptor (VEGFR)-mediated signaling. Finally, AgNP-treated tumors caused angiogenesis around tumors in B16F10 melanomas after they were injected into mice, and the Hb concentration in the tumors increased in a concentration-dependent manner with AgNP treatment. Thus, our study suggests that exposure to AgNPs can cause angiogenesis through the production of angiogenic factors.

Colorimetric detection of mutations in epidermal growth factor receptor using gold nanoparticle aggregation.

We have detected mutations in the epidermal growth factor receptor (EGFR) of non-small cell lung cancer cells using the selective aggregations of gold nanoparticles. Mutations in exon 19 and exon 21 of EGFR gene were detected in non-amplified genomic DNAs that were isolated from both the lung cancer cell lines and the cancer tissues of non-small cell lung cancer patients. At the optimal salt concentration, addition of the mutant DNA that was hybridized with the complementary probe into the suspension of unmodified gold nanoparticles caused the substantial aggregation of the gold nanoparticles and the color change of solution. Gold nanoparticles, however, did not exhibit significant aggregation, and the solution color remained unchanged with the addition of the wild type DNA that was hybridized with the probe. In the eight specimens from non-small cell lung cancer patients, we could detect the in-frame deletion mutant form in exon 19 and the L858R point mutation in exon 21 by selective aggregation with gold nanoparticles. These results were also confirmed by an independent direct sequencing method using a DNA analyzer. We found that selective aggregation with gold nanoparticles could be successfully applied to direct detection of EGFR mutations in non-amplified genomic DNAs.

Aggregation effects of gold nanoparticles for single-base mismatch detection in influenza A (H1N1) DNA sequences using fluorescence and Raman measurements.

Aggregation effects of gold nanoparticles (AuNPs) were examined for the discrimination of single point mutations through the hybridization of oligonucleotides (25-50 nM) modified with a fluorescent Texas red dye. The sequences of oligonucleotides were designed to detect the H1N1 virus gene. Single-base mismatch detection due to different adsorption propensities of oligonucleotides could be achieved using fluorescence quenching and surface-enhanced Raman scattering (SERS) properties of the dye. We observed that the addition of perfectly matched double stranded DNA (pmdsDNA), modified with the Texas red dye in the suspension of citrate-reduced AuNPs could increase fluorescence recovery intensities more substantially than either single-base mismatched double stranded DNA (sbmdsDNA) or single stranded DNA (ssDNA). We also tested DNA hybridization under both aggregation and near non-aggregation conditions for fluorescence measurements. A spectral difference in fluorescence intensity between pmdsDNA and sbmdsDNA appeared to be more discriminating under near non-aggregation than aggregation conditions. On the other hand, the SERS intensities of pmdsDNA and sbmdsDNA decreased more significantly than that of ssDNA under aggregation conditions, whereas we could not observe any SERS intensities under non-aggregation conditions.

Simultaneous detection of multiple mutations in epidermal growth factor receptor based on fluorescence quenching of quantum dots

We have developed a simultaneous detection method for two common mutations in the epidermal growth factor receptor gene based on the fluorescence quenching phenomenon caused by aggregation of CdSe quantum dots. For detection of the in-frame deletion in exon 19 and the L858R point mutation in exon 21, water-soluble CdSe quantum dots with two sizes were functionalized using four different types of probe oligonucleotides. Addition of target oligonucleotides with the deletion mutation in exon 19 into the suspensions caused crosslinking-induced aggregation of green-emitting quantum dots, followed by the fluorescence quenching while that with the L858R point mutation resulted in aggregation of yellow-emitting quantum dots. In addition, targets with both deletion and point mutations caused aggregation of both green- and yellow-emitting quantum dots. This method allows a simultaneous detection of mutations in exon 19 and 21 of EGFR gene in a single experiment. We found that minimum mutant concentration that could be detected by this method was as low as 2% for deletion mutation, and 5% for point mutation. PCR products of EGFR gene were also used to confirm that our method could be used to detect mutation in amplified DNA fragments.

Peptide nucleic acid-mediated aggregation of reduced graphene oxides and label-free detection of DNA mutation.

We have investigated peptide nucleic acid (PNA)-mediated aggregation of reduced graphene oxide (rGO) sheets. Addition of PNA into suspension of rGOs resulted in aggregation of rGOs, which could be easily detected with the naked eye. To elucidate the mechanism of rGO aggregation, we monitored the reaction by transmission electron microscopy, zeta potential measurement, and UV-vis spectroscopy. Our findings suggest that PNA adsorbed on the rGO surface and then acted as a cross-linker to induce aggregation. We also tested the effects of different nucleic acids on rGO aggregation and found that not only the single-stranded DNA, but also the PNA-DNA complex, could stabilize the suspension against aggregation through electrostatic repulsion. Based on our understanding on rGO aggregation, we attempted to detect mutations in epidermal growth factor receptor (EGFR) by designing PNA probe to be complementary to the mutant type DNA sequence. Our results showed that PNA-mediated rGO aggregation could successfully be used to detect mutations in EGFR with high selectivity and sensitivity.

Selective Energy Transfer Between Quantum Dots and Gold Nanoparticles for Detection of Multiple Mutations in Epidermal Growth Factor Receptor

Selective energy transfer between quantum dots and gold nanoparticles was used to simultaneously detect mutations in the epidermal growth factor receptor (EGFR) gene. We functionalized the surface of gold nanoparticles and green and red-emitting quantum dots using four different probe DNAs that were designed to be a perfect complementary to an in-frame deletion mutation in exon 19 or L858 R point mutation in exon 21 of EGFR. We found that the presence of the deletion mutation in exon 19 in target oligonucleotides caused fluorescence quenching at 525 nm due to energy transfer from green-emitting quantum dots to gold nanoparticles, whereas point mutation in exon 21 resulted in quenching at 620 nm due to energy transfer from red-emitting quantum dots to gold nanoparticles. This method could successfully be used to simultaneously detect the presence of two types of mutations in EGFR. We also defined a parameter (i.e., the extent of quenching) to quantify fluorescence quenching phenomenon. By varying the fraction of mutant type DNA in target oligonucleotides, we showed that detection sensitivity based on the extent of quenching was about 5%, which is lower than the conventional direct sequencing method.