Proteolysis-driven proliferation and rigidification of pepsin-resistant amyloid fibrils.

Proteolysis of amyloids is related to prevention and treatment of amyloidosis. What if the conditions for proteolysis were the same to those for amyloid formation? For example, pepsin, a gastric protease is activated in an acidic environment, which, interestingly, is also a condition that induces the amyloid formation. Here, we investigate the competition reactions between proteolysis and synthesis of amyloid under pepsin-activated conditions. The changes in the quantities and nanomechanical properties of amyloids after pepsin treatment were examined by fluorescence assay, circular dichroism and atomic force microscopy. We found that, in the case of pepsin-resistant amyloid, a secondary reaction can be accelerated, thereby proliferating amyloids. Moreover, after this reaction, the amyloid became 32.4 % thicker and 24.2 % stiffer than the original one. Our results suggest a new insight into the proteolysis-driven proliferation and rigidification of pepsin-resistant amyloids.

Colorimetric Nanoparticle-Embedded Hydrogels for a Biosensing Platform.

Hydrogels containing colorimetric nanoparticles have been used for ion sensing, glucose detection, and microbial metabolite analyses. In particular, the rapid chemical reaction owing to both the hydrogel form of water retention and the sensitive color change of nanoparticles enables the rapid detection of target substances. Despite this advantage, the poor dispersibility of nanoparticles and the mechanical strength of nanoparticle-hydrogel complexes have limited their application. In this study, we demonstrate a milliliter agarose gel containing homogeneously synthesized polyaniline nanoparticles (PAni-NPs), referred to as PAni-NP-hydrogel complexes (PNHCs). To fabricate the optimal PNHC, we tested various pH solvents based on distilled water and phosphate-buffered saline and studied the colorimetric response of the PNHC with thickness. The colorimetric response of the prepared PNHC to the changes in the pH of the solution demonstrated excellent linearity, suggesting the possibility of using PNHC as a pH sensor. In addition, it was verified that the PNHC could detect minute pH changes caused by the cancer cell metabolites without cytotoxicity. Furthermore, the PNHC can be stably maintained outside water for approximately 12 h without deformation, indicating that it can be used as a disposable patch-type wearable biosensing platform.

Quantification of doping state of redox sensitive nanoparticles for probing the invasiveness of cancer cells using surface enhanced Raman scattering.

Redox activity is known to regulate migration, invasion, metastasis, proliferation, and vascularization of cancer. Because cancer is heterogeneous, the role of redox activity in different cancers and cancer-related processes vary widely. In this study, water soluble, Tween 80-coated polyaniline (TPAni) nanoparticles were synthesized and used as nano-agents for sensing the redox activities of various cancer cells. To identify the relationship between the redox activity and the aggressiveness of cancer cells, two different cancer cell lines, derived from the same tissue but different with regards to aggressiveness, were selected for study. First, the cancer cell lines were incubated with TPAni nanoparticles, and an absorbance ratio obtained from the cell culture media was used as a colorimetric indicator of the redox activities of the cells. Simultaneously, hydrophobically modified filter papers coated with silver nanosnowflakes (SNSF) were used as sensing substrates for surface enhanced Raman scattering (SERS). SERS spectra obtained from varying concentrations of rhodamine 6G were used to confirm the detection limit of the SNSF-based SERS substrate. Cell culture media containing TPAni nanoparticles were treated with the SNSF-containing SERS substrates to examine the redox activities of the various cancer cell lines.The redox activities of cancer cell lines were confirmed by absorbance spectral analysis, and these redox activities were better identified via an SERS analysis method. A SNSF-containing SERS substrate, fabricated from SNSF and filter paper, was used to sense redox activity in cancer cell lines and to further identify correlations between redox activity and cancer cell line aggressiveness, as indicated by the use of EpCAM as a biomarker. Finally, potential of â€‹in vivo â€‹redox activity sensing was also confirmed.

Hand-Held Raman Spectrometer-Based Dual Detection of Creatinine and Cortisol in Human Sweat Using Silver Nanoflakes.

The conventional tissue biopsy method yields isolated snapshots of a narrow region. Therefore, it cannot facilitate comprehensive disease characterization and monitoring. Recently, the detection of tumor-derived components in body fluids─a practice known as liquid biopsy─has attracted increased attention from the biochemical research and clinical application viewpoints. In this vein, surface-enhanced Raman scattering (SERS) has been identified as one of the most powerful liquid-biopsy analysis techniques, owing to its high sensitivity and specificity. Moreover, it affords high-capacity spectral multiplexing for simultaneous target detection and a unique ability to obtain intrinsic biomolecule-fingerprint spectra. This paper presents the fabrication of silver nanosnowflakes (SNSFs) using the polyol method and their subsequent dropping onto a hydrophobic filter paper. The SERS substrate, which comprises the SNSFs and hydrophobic filter paper, facilitates the simultaneous detection of creatinine and cortisol in human sweat using a hand-held Raman spectrometer. The proposed SERS system affords Raman spectrometry to be performed on small sample volumes (2 µL) to identify the normal and at-risk creatinine and cortisol groups.

Active colorimetric lipid-coated polyaniline nanoparticles for redox state sensing in cancer cells.

Herein, lipid-coated polyaniline (LiPAni) nanoparticles were fabricated to monitor the redox state of cancer cells. To confirm the characteristics of LiPAni, we firstly analyzed the size and chemical structures of the LiPAni nanoparticles. The absorbance properties of the LiPAni nanoparticles were observed to vary with the pH conditions. Furthermore, cell viability tests conducted with breast cancer cell lines showed that the cell viability of the cells with LiPAni nanoparticles was dramatically increased compared to those with the Tween80-coated polyaniline nanoparticles (TPAni) as a control. Subsequently, the colors of the LiPAni nanoparticles were observed and analyzed using spectroscopic methods. Finally, in order to investigate the more accurate sensing of the redox state using the color changes of the LiPAni nanoparticles with cancer cell lines, dark field microscopic images and scattering spectra were recorded at the single nanoparticle scale. For the TPAni nanoparticles, there was only a change in brightness and no change in color, but for the LiPAni nanoparticles, there was a change of color from yellow to pink in the dark field images.

Multimodal cellular redox nanosensors based on self-doped polyaniline nanocomposites.

We have successfully fabricated a nanocomposite, which is composed of polyaniline (PAni) and pyrene butyric acid (Pyba) via a solvent shift method, which was self-doped at a neutral pH value. This PAni nanocomposite can act as a fine nanoagent expressing absorbance, fluorescence, and Raman properties according to the surrounding pH values.

Synthesis and Characterization of Water-Soluble Conjugated Oligoelectrolytes for Near-Infrared Fluorescence Biological Imaging.

Near-infrared (NIR) fluorophores attract increasing attention as a molecular marker (or probe) for in vivo and in vitro biological fluorescence imaging. Three types of new NIR fluorescent conjugated oligoelectrolytes (COEs: Q-FlTBTTFl, Q-FlBBTFl, and Q-FlTBBTTFl) are synthesized with quaternized ammonium ionic groups in their side-chains for water solubility. The emission wavelength is modulated in the range 600-1300 nm, by adjusting the intramolecular charge transfer in the molecular backbone based on the electron-rich fluorene (and/or thiophene) and electron-deficient benzo[2,1,3]thiadiazole (or benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole) moieties. The COEs show a remarkably larger Stokes shift (147-276 nm) compared to commercial rhodamine and cyanine dyes in water, avoiding self-quenching and interference from the excitation backscattered light. The photoluminescence (PL) quantum efficiency is improved substantially by up to 27.8% in water by fabricating a vesicular complex, COE/v, with a block ionomer, poly[(ethylene oxide)-block-(sodium 2-acrylamido-2-methyl-1-propanesulfonate)]. In vitro cellular uptake images with the COEs are obtained with good biocompatibility by confocal single-photon and two-photon microscopy. The ex vivo and in vivo images of a mouse xenograft model treated with the Q-FlBBTFl/v exhibit a substantially stronger fluorescence signal at the tumor site than at the other organs, highlighting the potential of the COE/v as an NIR fluorescent imaging agent for the diagnosis of cancer.

Biomarker-specific conjugated nanopolyplexes for the active coloring of stem-like cancer cells.

Stem-like cancer cells possess intrinsic features and their CD44 regulate redox balance in cancer cells to survive under stress conditions. Thus, we have fabricated biomarker-specific conjugated polyplexes using CD44-targetable hyaluronic acid and redox-sensible polyaniline based on a nanoemulsion method. For the most sensitive recognition of the cellular redox at a single nanoparticle scale, a nano-scattering spectrum imaging analyzer system was introduced. The conjugated polyplexes showed a specific targeting ability toward CD44-expressing cancer cells as well as a dramatic change in its color, which depended on the redox potential in the light-scattered images. Therefore, these polyaniline-based conjugated polyplexes as well as analytical processes that include light-scattering imaging and measurements of scattering spectra, clearly establish a systematic method for the detection and monitoring of cancer microenvironments.

Femto-molar detection of cancer marker-protein based on immuno-nanoplasmonics at single-nanoparticle scale.

We describe an in vitro biomarker sensor based on immuno-silver nanomarbles (iSNMs) and the nanoscattering spectrum imaging analysis system using localized surface plasmon resonance (LSPR). In particular, highly monodisperse SNMs with large figures of merit are prepared, and the sensing substrates are also fabricated using the nanoparticle adsorption method. The high sensitivity of the LSPR sensor based on an SNM is confirmed using various solvents that have different refractive indexes. For the sensitive and specific detection of epithelial cell adhesion molecules (EpCAMs) expressed on cancer cells, the surface of the SNM is conjugated with an anti-EpCAM aptamer, and molecular sensing for the EpCAM expression level is carried out using whole cell lysates from various cancer cell lines. Collectively, we have developed a biomarker-detectable LSPR sensor based on iSNMs, which allows for the sensitive and effective detection of EpCAMs at both the single-cell and femto-molar level.

Photothermal ablation of cancer cells using self-doped polyaniline nanoparticles.

Water-stable confined self-doping polyaniline nanocomplexes are successfully fabricated by nano-assembly using lauric acid both as a stabilizer and as a localized dopant. In particular, the colloidal stability of the polyaniline nanocomplexes in neutral pH and the photothermal potential by near-infrared light irradiation are characterized. We demonstrate that confined self-doping polyaniline nanocomplexes as a photothermal nanoagent are preserved in the doped state even at a neutral pH. Finally, confined self-doping polyaniline nanocomplexes aided by lauric acid are successfully applied for the photothermal ablation of cancer cells.

In vivo sensing of proteolytic activity with an NSET-based NIR fluorogenic nanosensor.

Biomedical in vivo sensing methods in the near-infrared (NIR) range, which that provide relatively high photon transparency, separation from auto-fluorescence background, and extended sensitivity, are being used increasingly for non-invasive mapping and monitoring of molecular events in cancer cells. In this study, we fabricated an NIR fluorogenic nanosensor based on the nanoparticle surface energy transfer effect, by conjugation of fluorescent proteolytic enzyme-specific cleavable peptides with gold nanorods (GNRs). Membrane-anchored membrane type 1-matrix metalloproteinases (MT1-MMPs), a family of zinc-dependent proteolytic enzymes, can induce the metastatic potential of cancer cells by promoting degradation of the extracellular matrix. Therefore, sensitive detection of MT1-MMP activity can provide essential information in the clinical setting. We have applied in vivo NIR sensing to evaluate MT1-MMP activity, as an NIR imaging target, in an MT1-MMP-expressing metastatic tumor mouse model.

A magnetic polyaniline nanohybrid for MR imaging and redox sensing of cancer cells.

A synthetic process for constructing an organo-metal nanohybrid is described. This process uses polyaniline as a ligand in order to fabricate magnetic nanoparticles. This nanohybrid shows imaging potential uses as a magnetic resonance imaging contrast agent and a redox-sensing probe simultaneously both in vitro and in vivo.

Correlation of VEGF genetic polymorphisms and lipid profile to aortic calcification.

BACKGROUND: Aortic calcification is developed due to accumulation of a large amount of calcium in the aorta of the heart and it is the leading cause of aortic valve replacement and third leading cause of cardiovascular disease. The purpose of this study was to investigate the relation between aortic calcification and VEGF SNPs (-2578C>A, -1154G>A and +936C>T) and to evaluate the association of these SNPs with biochemical parameter in relation to aortic calcification. METHODS: Aortic calcification was diagnosed by examining the posteroanterior chest X-rays by a radiologist and graded into four groups. The real-time polymerase chain reaction with melting curve analysis in LightCycler was used to genotype the VEGF SNPs. RESULTS: Among the VEGF SNPs, a significant genetic difference was found only between the aortic calcification and control group with VEGF SNP -2578C>A but haplotypes T-A-A of (+936/-1154/-2578) were significantly different in control and aortic calcification and could enhance the aortic calcification development. By regression analysis, it was found that age, hypertension, diabetes, dyslipidemia, and hyperhomocysteinemia were found significantly different with the different genotypes of VEGF SNPs which may induce aortic calcification development. CONCLUSION: Age, hypertension, diabetes, dyslipidemia, and hyperhomocysteinemia were established as aggravating factors for the aortic calcification in association with different VEGF genotypes.

Molecular recognition of proteolytic activity in metastatic cancer cells using fluorogenic gold nanoprobes.

We describe the development of biomarker-sensitive nanoprobes based on nanoparticle surface energy transfer (NSET) effect that enabling recognition of the expression of membrane type-1 matrix metalloproteinase (MT1-MMP) anchored on invasive cancer cells and its proteolytic activity simultaneously. First of all, we confirmed invasiveness of cancer cell lines (HT1080 and MCF7) via migration and invasion assay. We also prepared gold nanoparticle (GNP) acts as a quencher for fluorescein isothiocyanate (FITC). This FITC is conjugated in end-terminal of activatable fluorogenic peptide (ActFP). The ActFP attach to surface of GNP (GNP-ActFP) for a targeting moiety and proteolytic activity ligand toward MT1-MMP. The GNP-ActFP can generate fluorescence signal when ActFP is cleaved by proteolytic activity after targeting toward MT1-MMP. In order to study specificity for MT1-MMP, GNP-ActFP is treated to HT1080 and MCF7 cells, and then, we determine the in vitro targeting potential and fluorogenic activity of GNP-ActFP for MT1-MMP via fluorescence multi-reader. We also confirmed fluorogenic activity of GNP-ActFP via confocal microscopic imaging, and finally, endocytosis of GNP-ActFP is observed via cellular transmission electron microscopic imaging.

Localized surface plasmon resonance based nanobiosensor for biomarker detection of invasive cancer cells.

In this study, we describe the development of a cancer biomarker-sensitive nanobiosensor based on localized surface plasmon resonance that enables recognition for proteolytic activity of membrane type 1 matrix metalloproteinase (MT1-MMP) anchored on invasive cancer cells. First of all, we prepared biomarker-detectable substrate based on gold nanorods (GNRs) using nanoparticle adsorption method. The sensitivity of the sensing chip was confirmed using various solvents that have different refractive indexes. Subsequently, MT1-MMP-specific cleavable peptide was conjugated onto the surface of GNRs, and molecular sensing about proteolytic activity was conducted using MT1-MMP and cell lysates. Collectively, we developed a biomarker detectable sensor, which allows for the effective detection of proteolytic activity about MT1-MMP extracted from invasive cancer cells.

Aptamer-conjugated gold nanorod for photothermal ablation of epidermal growth factor receptor-overexpressed epithelial cancer.

Biomarker-specific photothermal nanoparticles that can efficiently sense markers that are overexpressed in distinguished adenocarcinomas have attracted much interest in an aspect of efficacy increase of cancer treatment. We demonstrated a promising prospect of a smart photothermal therapy agent employing anti-epidermal growth factor receptor aptamer (AptEGFR)-conjugated polyethylene glycol (PEG) layted gold nanorods (AptEGFR-PGNRs). The cetyltrimethylammonium bromide bilayer on GNRs was replaced with heterobifunctional PEG (COOH-PEG-SH) not only to serve as a biocompatible stabilizer and but also to conjugate AptEGFR. Subsequently, to direct photothermal therapy agent toward epithelial cancer cells, the carboxylated PEGylated GNRs (PGNRs) were further functionalized with AptEGFR using carbodiimide chemistry. Then, to assess the potential as biomarker-specific photothermal therapy agent of synthesized AptEGFR-PGNRs, the optical properties, biocompatibility, colloidal stability, binding affinity, and epicellial cancer cell killing efficacy in vitro/in vivo under near-infrared laser irradiation were investigated. As a result, AptEGFR-PGNRs exhibit excellent tumor targeting ability and feasibility of effective photothermal ablation cancer therapy.

CD44-specific supramolecular hydrogels for fluorescence molecular imaging of stem-like gastric cancer cells.

We describe a near-infrared-sensitive molecular imaging probe based on hydrogel complexes that can target a stem-like gastric cancer cell marker (CD44, a marker that often correlates with a poor prognosis in patients). Thus, CD44-targetable and near-infrared-sensitive supramolecular hydrogels (NIRSHs, Cy5.5-conjugated polyethyleneimine/hyaluronic acid polyplexes) were fabricated by polyplexing in an aqueous medium. NIRSHs demonstrated good water-stability, biocompatibility, and specificity to CD44-expressing stem-like gastric cancer cells. Furthermore, NIR-sensitive in vivo imaging potentials of CD44-targetable NIRSHs for heterotopic/orthotopic xenograft mouse models were investigated.

Highly selective CD44-specific gold nanorods for photothermal ablation of tumorigenic subpopulations generated in MCF7 mammospheres.

Heterogeneous stem-like populations within tumor tissues are the primary suspects in causing cancer recurrence and malignancy. It is essential to selectively kill these tumorigenic populations. We created a novel system for photothermally ablating specific cells from three-dimensional mammospheres. A CD44-positive subpopulation, with tumorigenic and self-renewal potential, spontaneously arises in MCF7 breast cancer cell-engineered mammospheres. Using anti-CD44 antibody-linked gold nanorods, which strongly absorb near infrared light and increase local temperature, we effectively targeted and photo-ablated atypical cells. This biomarker-specific photothermal ablation model, using a smart nanoplatform, is a promising new strategy for selectively killing cancer cells, while sparing normal tissues.