Efficient targeted pDNA/siRNA delivery with folate-low-molecular-weight polyethyleneimine-modified pullulan as non-viral carrier.

Folate receptor (FR)-mediated gene/short interfering RNA (siRNA) targeting shows advantage for the delivery of gene/siRNA into specific FR-overexpressing cancer cells. In this study, the non-targeted gene vector P-PEI was synthesized by grafting low-molecular-weight (1kDa) branched polyethyleneimine (PEI) to succinylated pullulan, and the targeted gene vector P-PEI-FA was synthesized by coupling the carboxyl of folate (FA) to the amino of PEI. Gel electrophoresis retardation assay demonstrated that both P-PEI and P-PEI-FA can efficiently wrap pDNA and siRNA with electrostatic interaction at N/P ratios higher than 1.56 and can protect pDNA from degradation by DNase I and serum. Compared with PEI/pDNA, P-PEI/pDNA and P-PEI-FA/pDNA showed lower cytotoxicity against different cells. Under serum-containing conditions, compared with Lipofamine 2000/DNA and Lipofamine2000/siRNA, P-PEI-FA/DNA at N/P ratio of 6.25 displayed higher gene transfection efficiency, whereas P-PEI-FA/siRNA at N/P ratio of 12.5 demonstrated better enhanced gene silencing effect. P-PEI-FA/siRNA can also deliver FAM-labeled siRNA to endosomes and escape. Moreover, the gene transfection and silencing effects of P-PEI-FA were higher than those of P-PEI, and were dependent on the dose of FA in FR(+) HeLa cells. Thus, P-PEI-FA can assist DNA or siRNA targeting to FR-overexpressing cells, and the uptake pathway of P-PEI-FA/siRNA was FR-mediated endocytosis. These results indicate that P-PEI-FA is a potential candidate for safe and targeted gene delivery applications.

Fluorescence discrimination of cancer from inflammation by molecular response to COX-2 enzymes.

Accurate identification of cancer from inflammation and normal tissue in a rapid, sensitive, and quantitative fashion is important for cancer diagnosis and resection during surgery. Here we report the use of cyclooxygenase-2 as a marker for identification of cancer from inflammation and the design of a novel smart COX-2-specific fluorogenic probe (NANQ-IMC6). The probe's fluorescence is "turned on" in both inflammations and cancers where COX-2 is overexpressed. Intriguingly, the fluorescent emission is quite different at these two sites with different expression level of COX-2. Hence, NANQ-IMC6 can not only distinguish normal cells/tissues from cancer cells/tissues but also distinguish the latter from sites of inflammation lesions by the different fluorescence recognition of NANQ-IMC6 for COX-2 enzymes. Following spraying with the NANQ-IMC6 solution, cancerous tissue, inflamed tissues, and normal tissues can be accurately discriminated in vivo by the unaided eye using a hand-held ultraviolet lamp emitting at 365 nm. So the probe may have potential application varying from cancer inflammation diagnosis to guiding tumor resection during surgery.

An off-on COX-2-specific fluorescent probe: targeting the Golgi apparatus of cancer cells.

Identifying cancer cells and quantifying cancer-related events in particular organelles in a rapid and sensitive fashion are important for early diagnosis and for studies on pathology and therapeutics of cancers. Herein a smart "off-on" cyclooxygenase-2-specific fluorescence probe (ANQ-IMC-6), able to report the presence of cancer cells and to image Golgi-related events, has been designed and evaluated. Cyclooxygenase-2 (COX-2) has been used as imaging target in the probe design, since this enzyme is a biomarker of virtually all cancer cell lines. In the free state in aqueous solution, ANQ-IMC-6 mainly exists in a folded conformation where probe fluorescence is quenched through photoinduced electron transfer between the fluorophore acenaphtho[1,2-b]quinoxaline (ANQ) and the recognition group, indomethacin (IMC). Fluorescence is turned on, by restraining the photoinduced electron transfer, when ANQ-IMC-6 is forced to adopt the unfolded state following binding to COX-2 in the Golgi apparatus of cancer cells. ANQ-IMC-6 provides high signal-to-background staining and has been successfully used to rapidly differentiate cancer cells from normal cells when using flow cytometry and one- and two-photon fluorescence microscopic imaging. Furthermore, ANQ-IMC-6 may be able to visualize dynamic changes of the Golgi apparatus during cancer cell apoptosis, with possible application to early diagnosis.

A BODIPY-based fluorescent dye for mitochondria in living cells, with low cytotoxicity and high photostability.

A BODIPY-based dye, OBEP, has been developed to act as a mitochondrial fluorescence probe. This dye is of high stability, low toxicity and insensitive in a pH range as wide as pH 2-10. Its uptake into mitochondria is independent of mitochondrial membrane potential in living cells. OBEP can label swollen mitochondria resulting from different degrees of cell damage in light and resist fading even after 12 h of incubation.

Zn(2+) binding-enabled excited state intramolecular proton transfer: a step toward new near-infrared fluorescent probes for imaging applications.

In order to facilitate the in vivo study of zinc-related biology, it is essential to develop a zinc-selective sensor that exhibits both near-infrared (NIR) emission and larger Stokes shift. A fluorescent sensor, Zinhbo-5, has been constructed by using bis(benzoxazole) ligand with 2, 2'-dipicolylamine (DPA) as metal ion receptor. In aqueous solution, Zinhbo-5 exhibits high sensitivity (K(d) = 2.58 nM(2) ) and selectivity for Zn(2+) cation, revealing about 14-fold fluorescence enhancement upon zinc binding to give green emission. Remarkably, Zn(2+) binding to Zinhbo-5 switches on the excited state intramolecular proton transfer (ESIPT), producing the desirable near-infrared region (over 710 nm) with large Stokes shift (ca. 240 nm). The new probe is demonstrated to be useful for in vivo imaging of the intracellular Zn(2+) ion. The Zinhbo-5 is also useful for detecting zinc ion distribution during the development of living zebrafish embryos.

FRET spectral unmixing: a ratiometric fluorescent nanoprobe for hypochlorite.

A specific ratiometric nanoprobe for hypochlorite was constructed as a paradigm of FRET spectral unmixing. The separation of FRET pairs' emissions reaches 175 nm, which ensures that the FRET probing is more accurate. This new nanoprobe shows high selectivity and potential in biological systems.

Vitamin E renders protection to PC12 cells against oxidative damage and apoptosis induced by single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWCNTs) are potential candidates in many biomedical applications. However, many reports demonstrated its potential toxicity to human and other biological systems. Our study has demonstrated that SWCNTs can induce apoptosis and oxidative damage on PC12 cells, an in vitro model of neuronal cells. In the present study, we for the first time investigated the neuroprotective effects of vitamin E (VE) on SWCNT-induced neurotoxicity in cultured PC12 cells. Vitamin E (0.01-2mM) increased PC12 cells viability and significantly attenuated SWCNTs-induced apoptotic cell death in a time and dose-dependent manner, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release and morphological observation. The presence of VE inhibited the formation of reactive oxygen species (ROS), decreased the level of lipid peroxide, elevated the level of glutathione (GSH) and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Additionally, VE blocked the reduction in the mitochondrial membrane potential and the activation of caspase-3. VE prevented the down-regulation of Bcl-2 expression and up-regulation of Bax expression induced by SWCNTs in PC12 cells. In summary, VE might protect PC12 cells from the injury induced by SWCNTs through the down-regulation of oxidative stress and prevention of mitochondrial-mediated apoptosis.