No abstract

Spatiotemporal control of singlet oxygen ((1)O2) release is a major challenge for photodynamic therapy (PDT) against cancer with high therapeutic efficacy and minimum side effects. Here a selenium-rubyrin (NMe2Se4N2)-loaded nanoparticle functionalized with folate (FA) was designed and synthesized as an acidic pH-activatable targeted photosensitizer. The nanoparticles could specifically recognize cancer cells via the FA-FA receptor binding and were selectively taken up by cancer cells via receptor-mediated endocytosis to enter lysosomes, in which NMe2Se4N2 was activated to produce (1)O2. The pH-controllable release of (1)O2 specially damaged the lysosomes and thus killed cancer cells in a lysosome-associated pathway. The introduction of selenium into the rubyrin core enhanced the (1)O2 generation efficiency due to the heavy atom effect, and the substitution of dimethylaminophenyl moiety at meso-position led to the pH-controllable activation of NMe2Se4N2. Under near-infrared (NIR) irradiation, NMe2Se4N2 possessed high singlet oxygen quantum yield (ΦΔ) at an acidic pH (ΦΔ = 0.69 at pH 5.0 at 635 nm) and could be deactivated at physiological pH (ΦΔ = 0.06 at pH 7.4 at 635 nm). The subcellular location-confined pH-activatable photosensitization at NIR region and the cancer cell-targeting feature led to excellent capability to selectively kill cancer cells and prevent the damage to normal cells, which greatly lowered the side effects. Through intravenous injection of FA-NMe2Se4N2 nanoparticles in tumor-bearing mice, tumor elimination was observed after NIR irradiation. This work presents a new paradigm for specific PDT against cancer and provides a new avenue for preparation of highly efficient photosensitizers.

This work designs a telomerase-responsive mesoporous silica nanoparticle (MSN) to realize in situ "off-on" imaging of intracellular telomerase activity. In the wrapping DNA (O1) sealed MSN probe, a black hole fluorescence quencher is covalently immobilized on the inner walls of the mesopores, while fluorescein is loaded in the mesopores. In the presence of telomerase and dNTPs, the designed O1 can be extended and then moves away from the MSN surface via forming a rigid hairpin-like DNA structure. Thus the O1 can act as a "biogate" to block and release fluorescein for "off-on" switchable fluorescent imaging. The MSN probe exhibits good performance for sensitive in situ tracking of telomerase activity in living cells. The practicality of this protocol has been verified by monitoring the change of cellular telomerase activity in response to telomerase-related drugs.

Phosphorylation is the most common posttranslational modification of the ␣-crystallins in the human lens. These phosphorylated forms are not only important because of their abundance in aging lenses and the implications for cataract but also because they have been identified in patients with degenerative brain disease. By using mimics corresponding to the reported in vivo phosphorylation sites in the human lens, we have examined the effects of phosphorylation upon the chaperone-like properties and structure of ␣B-crystallin. Here we show that phosphorylation of ␣B-crystallin at Ser-45 results in uncontrolled aggregation. By using an innovative tandem mass spectrometry approach, we demonstrate how this alteration in behavior stems from disruption of dimeric substructure within the polydisperse ␣B-crystallin assembly. This structural perturbation appears to disturb the housekeeping role of ␣B-crystallin and consequently has important implications for the disease states caused by protein aggregation in the lens and deposition in non-lenticular tissue.The mammalian small heat shock protein (sHSP) 1 ␣B-crystallin, although systemically expressed (1), is found primarily in the eye lens where it associates with the closely related ␣A-crystallin into large hetero-oligomers. While its lenticular function is also structural, both ␣B-and ␣A-crystallin and the hetero-oligomer ␣-crystallin (2) have been shown to display molecular chaperone activity in vitro and to arrest the aggregation of the ␤-and ␥-crystallins in the lens (3). Unlike ATP-dependent chaperones, the ␣-crystallins and other sHSPs are not thought to actively refold non-native proteins but rather to incorporate them into large complexes, thereby preventing their nonspecific aggregation (4 -6). This property of the ␣-crystallins is regarded as crucial in the maintenance of lens transparency.As there is no protein turnover in the central part of the lens, the exceptionally long lifetime of lenticular proteins means that the ␣-crystallins are susceptible to the accumulation of a variety of posttranslational modifications that are thought to disrupt their structure (7-9). For ␣B-crystallin in the lens, the major modifications have been identified as phosphorylation at serine residues 19, 45, and 59 (10). Despite phosphorylation being a common feature of ␣B-crystallin both inside and outside the lens (11), the in vivo significance of these modifications and their effect on chaperone-like activity remain unclear (12). Phosphorylation of ␣-crystallin has been reported variously to have no effect on chaperone activity (13,14), to reduce chaperone activity (15), and to cause a decrease in oligomeric size (16). Furthermore, it has been shown that in vitro phosphorylation of ␣B-crystallin compromises its inhibitory activity toward actin polymerization (17). The use of site-directed mutagenic substitution of aspartate for serine residues to mimic phosphorylated ␣B-crystallin led to the report of smaller oligomers with significantly reduced chaperone efficacy (18).␣B-Crysta...

A trifunctional photosensitizer was designed to achieve highly selective near-infrared tumor imaging, efficient photodynamic therapy and therapeutic self-monitoring.

alpha-Crystallin is a major lens protein, comprising up to 40% of total lens proteins, where its structural function is to assist in maintaining the proper refractive index in the lens. In addition to its structural role, it has been shown to function in a chaperone-like manner. The chaperone-like function of alpha-crystallin will help prevent the formation of large light-scattering aggregates and possibly cataract. In the lens, alpha-crystallin is a polydisperse molecule consisting of a 3:1 ratio of alpha A to alpha B subunits. In this study, we expressed recombinant alpha A- and alpha B-crystallin in E. coli and compared the polydispersity, structure and aggregation state between each other and native bovine lens alpha-crystallin. Using gel permeation chromatography to assay for polydispersity, we found native alpha-crystallin to be significantly more polydisperse than either recombinant alpha A- or alpha B-crystallin, with alpha B-crystallin having the most homogeneous structure of the three. Reconstructed images of alpha B-crystallin obtained with cryo-electron microscopy support the concept that alpha B-crystallin is an extremely dynamic molecule and demonstrated that it has a hollow interior. Interestingly, we present evidence that native alpha-crystallin is significantly more thermally stable than either alpha A- or alpha B-crystallin alone. In fact, our experiments suggest that a 3:1 ratio of alpha A to alpha B subunit composition in an alpha-crystallin molecule is optimal in terms of thermal stability. This fascinating result explains the stoichiometric ratios of alpha A- and alpha B-crystallin subunits in the mammalian lens.

A nicked molecular beacon (MB)-functionalized probe has been designed for in situ imaging and detection of intracellular telomerase activity. The nick separates the MB into two segments: a shorter telomerase primer (TSP) sequence as a part of the 5'-end stem and a longer sequence to form a loop with one thiol-labeled 3'-end stem. The MB can be opened by substitutional hybridization of the telomerase-triggered stem elongation product, which leads to separation of the Cy5 at the 5'-end nick from the gold nanoparticle (AuNP) as the nanocarrier and thus inhibits the energy transfer from Cy5 to AuNP. Upon endocytosis of the probe, the TSP can be extended by intracellular telomerase at its 3' end to produce the telomeric repeated sequence, which leads to the inner chain substitution and thus turns on the fluorescence of Cy5. The probe provides a one-step incubation technique for quantification and monitoring of the telomerase activity in living cells. The practicality of the proposed approach for distinguishing tumor cells from normal cells and monitoring the decrease of telomerase activity during treatment with antitumor drugs demonstrates its potential in clinical diagnostic and therapeutic monitoring.

No abstract