Cornelia Man

Polymer-coated NaYF4: Yb3+, Er3+ upconversion nanoparticles for charge-dependent cellular imaging

Lanthanide-doped upconversion nanoparticles (UCNPs) are considered promising novel near-infrared (NIR) bioimaging agents with the characteristics of high contrast and high penetration depth. However, the interactions between charged UCNPs and mammalian cells have not been thoroughly studied, and the corresponding intracellular uptake pathways remain unclear. Herein, our research work involved the use of a hydrothermal method to synthesize polyvinylpyrrolidone-coated UCNPs (UCNP-PVP), and then a ligand exchange reaction was performed on UCNP-PVP, with the help of polyethylenimine (PEI) and poly(acrylic acid) (PAA), to generate UCNP-PEI and UCNP-PAA. These polymer-coated UCNPs demonstrated good dispersibility in aqueous medium, had the same elemental composition and crystal phase, shared similar TEM and dynamic light scattering (DLS) size distribution, and exhibited similar upconversion luminescence efficiency. However, the positively charged UCNP-PEI evinced greatly enhanced cellular uptake in comparison with its neutral or negative counterparts, as shown by multiphoton confocal microscopy and inductively coupled plasma mass spectrometry (ICP-MS) measurements. Meanwhile, we found that cationic UCNP-PEI can be effectively internalized mainly through the clathrin endocytic mechanism, as revealed by colocalization, chemical, and genetic inhibitor studies. This study elucidates the role of the surface polymer coatings in governing UCNP-cell interactions, and it is the first report on the endocytic mechanism of positively charged lanthanide-doped UCNPs. Furthermore, this study provides important guidance for the development of UCNPs as specific intracellular nanoprobes, allowing us to control the UCNP-cell interactions by tuning surface properties.

Gold-doxorubicin nanoconjugates for overcoming multidrug resistance

UNLABELLED Multidrug resistance (MDR) is a major clinical obstacle to the success of cancer chemotherapy. Here we developed a gold-doxorubicin (DOX) nanoconjugates system to overcome MDR. Gold nanoparticles (AuNPs) were first PEGylated as Au-PEG-NH(2), and DOX was then grafted onto AuNPs via a cleavable disulfide linkage (Au-PEG-SS-DOX). Confocal images revealed that the extent of intracellular uptake of Au-PEG-SS-DOX was greater than that of free DOX in the MDR cells, and inductively coupled plasma mass spectroscopy analysis further confirmed that AuNPs significantly increased the level of drug accumulation in MDR cells at a nanoparticles dose greater than 15 μM. The cytotoxicity study demonstrated that the Au-PEG-SS-DOX nanoconjugates system efficiently released the anticancer drug DOX and enhanced its cytotoxicity against MDR cancer cells. This study highlights the potential of using AuNPs for overcoming of MDR in cancer chemotherapy. FROM THE CLINICAL EDITOR This study demonstrates that gold nanoparticles can be successfully applied to overcome MDR in cancer chemotherapy.

Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase‐immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16INK4A locus and downregulation of RASSF1A expression. The deletion of the p16INK4A locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array‐based CGH revealed a gain of a 17q21‐q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16INK4A locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF‐κB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development.

Emissive Terbium Probe for Multiphoton in Vitro Cell Imaging

A polymeric terbium complex that can be excited by near-infrared excitation at 800 nm via multiphoton absorption processes has been synthesized. This complex has been demonstrated to show strong, observable, three-photon-induced f-f emission in cell imaging. In vitro studies carried out in three carcinoma cell lines (A549, HONE1, and HeLa) have been performed and shown to have low cytotoxicity. This complex is therefore a potential candidate for future infrared excitation imaging dyes.

Efficient Immortalization of Primary Nasopharyngeal Epithelial Cells for EBV Infection Study

Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma.

A bioaccumulative cyclometalated platinum(II) complex with two-photon-induced emission for live cell imaging

The cyclometalated platinum(II) complex [Pt(L)Cl], where HL is a new cyclometalating ligand 2-phenyl-6-(1H-pyrazol-3-yl)pyridine containing C(phenyl), N(pyridyl), and N(pyrazolyl) donor moieties, was found to possess two-photon-induced luminescent properties. The two-photon-absorption cross section of the complex in N,N-dimethylformamide at room temperature was measured to be 20.8 GM. Upon two-photon excitation at 730 nm from a Ti:sapphire laser, bright-green emission was observed. Besides its two-photon-induced luminescent properties, [Pt(L)Cl] was able to be rapidly accumulated in live HeLa and NIH3T3 cells. The two-photon-induced luminescence of the complex was retained after live cell internalization and can be observed by two-photon confocal microscopy. Its bioaccumulation properties enabled time-lapse imaging of the internalization process of the dye into living cells. Cytotoxicity of [Pt(L)Cl] to both tested cell lines was low, according to MTT assays, even at loadings as high as 20 times the dose concentration for imaging for 6 h.

Surface decoration of selenium nanoparticles by mushroom polysaccharides–protein complexes to achieve enhanced cellular uptake and antiproliferative activity

By using mushroom polysaccharides–protein complexes (PSP) as the capping agent, size controllable and highly stable selenium nanoparticles (SeNPs) have been successfully created in a simple redox system of sodium selenite and ascorbic acid. SeNPs were capped with PSP through strong physical adsorption of hydroxyl groups of polysaccharides and imino groups of proteins on the surface of SeNPs. PSP surface decoration significantly enhanced the cellular uptake of SeNPs through endocytosis. Treatment with PSP–SeNPs significantly inhibited the growth of MCF-7 human breast cacinoma cells through induction of apoptosis with the involvement of PARP cleavage and caspase activation. Moreover, PSP–SeNPs not only significantly induced dose-dependent disruption of mitochondrial membrane potential in MCF-7 cells after 24 h treatment, but it also enhanced reactive oxygen species (ROS) generation as early as 15 min, indicating that ROS-mediated mitochondrial dysfunction may play an important role in PSP–SeNPs-induced apoptosis. Our results suggest that PSP–SeNPs may be a candidate for further evaluation as a chemopreventive agent for human cancers, and the strategy to use PSP as a surface decorator could be a highly efficient way to enhance the cellular uptake and anticancer efficacy of nanomaterials.

An amphiphilic ruthenium(II)-polypyridyl appended porphyrin as potential bifunctional two-photon tumor-imaging and photodynamic therapeutic agent.

An amphiphilic porphyrin appended with a Ru(II)-polypyridyl complex (Ru-P) showing a moderate two-photon absorption cross-section (178.0+/-26.8GM), high singlet oxygen quantum yield and rapid cellular uptake was synthesized. In vitro study using human nasopharyngeal carcinoma cells showed that Ru-P exhibited a strong two-photon induced fluorescence upon uptake, lysosomal localization and potent two-photon induced cytotoxicity. These results show that Ru-P, which was designed to enhance its cellular uptake, can potentially be used as an efficacious bifunctional two-photon tumor-imaging and photodynamic therapeutic agent despite its moderate two-photon absorption cross-section.

Two-Photon Plasma Membrane Imaging in Live Cells by an Amphiphilic, Water-Soluble Cyctometalated Platinum(II) Complex

An amphiphilic, water-soluble cyclometalated Pt(II) complex with two-photon emission properties has been developed as a molecular marker specific for in vitro plasma membrane staining.

Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells

Epstein–Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and can be detected in early premalignant lesions of nasopharyngeal epithelium. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by the EBV and is believed to play a role in transforming premalignant nasopharyngeal epithelial cells into cancer cells. RASSF1A is a tumor-suppressor gene commonly inactivated in many types of human cancer including NPC. In this study, we report a novel function of LMP1, in down-regulating RASSF1A expression in human epithelial cells. Downregulation of RASSF1A expression by LMP1 is dependent on the activation of intracellular signaling of NF-κB involving the C-terminal activating regions (CTARs) of LMP1. LMP1 expression also suppresses the transcriptional activity of the RASSF1A core promoter. RASSF1A stabilizes microtubules and regulates mitotic events. Aberrant mitotic spindles and chromosome aberrations are reported phenotypes in RASSF1A inactivated cells. In this study, we observed that LMP1 expression in human epithelial cells could induce aberrant mitotic spindles, disorganized interphase microtubules and aneuploidy. LMP1 expression could also suppress microtubule dynamics as exemplified by tracking movements of the growing tips of microtubules in live cells by transfecting EGFP-tagged EB1 into cells. The aberrant mitotic spindles and interphase microtubule organization induced by LMP1 could be rescued by transfecting RASSF1A expression plasmid into cells. Downregulation of RASSF1A expression by LMP1 may facilitate its role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells.