See-Lok Ho

Effect of surface-functionalized nanoparticles on the elongation phase of beta-amyloid (1-40) fibrillogenesis.

The influence of nanoparticles of various sizes and surface functionalities on the self-assembling fibrillogenesis of beta-amyloid (1-40) peptide was investigated. Functionalized nanoparticles including quantum dots and gold nanoparticles were co-incubated with monomeric Aβ(1-40) peptides under seed-mediated growth method to study their influences on the elongation phase of the fibrillogenesis. It is observed that charge-to-surface area ratio of the nanoparticles and the functional moiety and electrostatic charges of the conjugated ligands on the particle surfaces took crucial regulatory role in the Aβ(1-40) fibrillogenesis.

Inhibition of β-Amyloid Aggregation by Albiflorin, Aloeemodin and Neohesperidin and their Neuroprotective Effect on Primary Hippocampal Cells Against β-Amyloid Induced Toxicity

Being one of the hallmarks of Alzheimers disease, β-amyloid (Aβ) aggregates induce complicated neurotoxicity. Evidences show that the underlying mechanism of neurotoxicity involves a glutamate receptor subtype, N-methyl-D-aspartate (NMDA) receptor, an increase in intracellular calcium(II) ion loading as well as an elevation in oxidation stress. In this work, among the 35 chemical components of Chinese herbal medicines (CHMs) being screened for inhibitors of Aβ aggregation, four of them, namely albiflorin, aloeemodin, neohesperidin and physcion, were found for the first time to exhibit a potent inhibitory effect on Aβ(1-40) and Aβ(1-42) aggregation. Their neuroprotective capability on primary hippocampal neuronal cells was also investigated by MTT assay, ROS assay and intracellular calcium(II) ion concentration measurement. It was interesting to find that physcion was rather toxic to neuronal cells while albiflorin, aloeemodin and neohesperidin reduced the toxicity and ROS induced by both monomeric and oligomeric Aβ species. In addition, albiflorin was particularly powerful in maintaining the intracellular Ca(2+) concentration.

Self-assembling protein platform for direct quantification of circulating microRNAs in serum with total internal reflection fluorescence microscopy

MicroRNA (miRNA) has recently emerged as a new and important class of cellular regulators. Strong evidences showed that aberrant expression of miRNA is associated with a broad spectrum of human diseases, such as cancer, diabetes, cardiovascular and psychological disorders. However, the short length and low abundance of miRNA place great challenges for conventional techniques in the miRNA quantification and expression profiling. Here, we report a direct, specific and highly sensitive yet simple detection assay for miRNA without sample amplification. A self-assembled protein nanofibril acted as an online pre-concentrating sensor to detect the target miRNA. Locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1 pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patients serums was also demonstrated.

Direct quantification of circulating miRNAs in different stages of nasopharyngeal cancerous serum samples in single molecule level with total internal reflection fluorescence microscopy.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate human gene expression at the post-transcriptional level. Growing evidence indicates that the expression profile of miRNAs is highly correlated with the occurrence of human diseases including cancers. Playing important roles in complex gene regulation processes, the aberrant expression pattern of various miRNAs is implicated in different types and even stages of cancer. Besides localizing in cells, many of these miRNAs are found circulating around the body in a wide variety of fluids such as urine, serum and saliva. Surprisingly, these extracellular circulating miRNAs are highly stable and resistant to degradation, and therefore, are considered as promising biomarkers for early cancer diagnostic via noninvasive extraction from body fluids. Unfortunately, the abundance of these small RNAs is ultralow in the body fluids, making it challenging to quantify them in complex sample matrixes. Establishing a sensitive, specific yet simple assay for an accurate quantification of circulating miRNAs is therefore desirable. Our group previously reported a sensitive and specific detection assay of miRNAs in single molecule level with the aid of total internal reflection fluorescence microscopy. In this work, we advanced the assay to differentiate the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, a locked nucleic acid-modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 μL of serum and the whole assay can be completed in 1 h. The experimental results agreed well with those previously reported whereas the quantity of miR-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising noninvasive detection tool for early NPC diagnosis, monitoring and staging.

Dual-Modal NIR-Fluorophore Conjugated Magnetic Nanoparticle for Imaging Amyloid-β Species In Vivo

Senile plaques, the extracellular deposit of amyloid-β (Aβ) peptides, are one of the neuropathological hallmarks found in Alzheimers disease (AD) brain. The current method of brain imaging of amyloid plaques based on positron emission tomography (PET) is expensive and invasive with low spatial resolution. Thus, the development of sensitive and nonradiative amyloid-β (Aβ)-specific contrast agents is highly important and beneficial to achieve early AD detection, monitor the disease progression, and evaluate the effectiveness of potential AD drugs. Here a neuroprotective dual-modal nanoprobe developed by integrating highly Aβ-specific and turn-on fluorescence cyanine sensors with superparamagnetic iron oxide nanoparticles as an effective near-infrared imaging (NIRI)/magnetic resonance imaging (MRI) contrast agent for imaging of Aβ species in vivo is reported. This Aβ-specific probe is found not only nontoxic and noninvasive, but also highly blood brain barrier permeable. It also shows a potent neuroprotective effect against Aβ-induced toxicities. This nanoprobe is successfully applied for in vivo fluorescence imaging with high sensitivity and selectivity to Aβ species, and MRI with high spatial resolution in an APP/PS1 transgenic mice model. Its potential as a powerful in vivo dual-modal imaging tool for early detection and diagnosis of AD in humans is affirmed.

Effective Theranostic Cyanine for Imaging of Amyloid Species in Vivo and Cognitive Improvements in Mouse Model

We report herein an investigation of carbazole-based cyanine, (E)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)-vinyl)-1-methyl-quinolin-1-iumiodide (SLM), as an effective theranostic agent for Alzheimer’s disease (AD). This cyanine exhibited desirable multifunctional and biological properties, including amyloid-β (Aβ)-oligomerization inhibition, blood–brain barrier permeability, low neurotoxicity, neuroprotective effect against Aβ-induced toxicities, high selectivity and strong binding interactions with Aβ peptide/species, good biostability, as well as strong fluorescence enhancement upon binding to Aβ species for diagnosis and therapy of AD. This cyanine has been successfully applied to perform near-infrared in vivo imaging of Aβ species in transgenic AD mouse model. The triple transgenic AD mice intraperitoneally treated with SLM showed significant recovery of cognitive deficits. Furthermore, those SLM-treated mice exhibited a substantial decrease in both of oligomeric Aβ contents and tau proteins in their brain, which was attributed to the induction of autophagic flux. These findings demonstrated for the first time that SLM is an effective theranostic agent with in vivo efficacy for diagnosis and treatment of AD in mouse models.

NEAR-INFRARED IMAGING OF β-AMYLOID SPECIES/PLAQUES IN ANIMAL MODEL

at age of 20-month-old were used. We simultaneously injected Shiga-Y5 and Shiga-X22 at a dose of 200 mg/kg into the tail vein of three mice. At 3-h after the injection, F chemical shift imaging was obtained using a 7-tesla MR scanner. Another three mice were killed by overdose of sodium pentobarbital and we removed the brain. Then, the concentrations of Ab oligomers were measured by enzyme-linked immunosorbent assay (ELISA). Results: The F MR signal of Shiga-Y5 appeared mainly in the olfactory bulb, the subcortical area, and the cerebellum. In contrast, the F MR signal of Shiga-X22 displayed predominantly in the olfactory bulb, the cortex, and the cerebellum. Then, we produced a subtracted image by subtracting F MR images of Shiga-X22 from that of Shiga-Y5. F MR signal in the subtracted image was located in the subcortical area. ELISA measurement showed that Ab oligomers uniformly distributed in the brain except for the brainstem, and there is no specific region showing the accumulation of Ab oligomers. In contrast, the level of insoluble Ab was significantly less in the subcortical area where the concentration of insoluble Ab was relatively low. The imbalance between relative high level of Ab oligomers and the less level of insoluble Ab in the subcortical area may contribute to intense F MR signal in the subtracted image. Conclusions:We simultaneously injected Shiga-Y5 and Shiga-X22 into the tail vein of APPswe/ PS1dE9 double transgenic mice, and then obtained an image in the brain by subtracting FMR images of Shiga-X22 from those of Shiga-Y5 to reflect the distribution of Ab oligomers.