Thermo- and pH-sensitive nanoparticles of poly (N-isopropylacrylamide-decenoic acid-1-vinylimidazole) for ultrasound switchable fluorescence imaging.

We herein report the synthesis of poly (9-decenoic acid-1-vinylimidazole-N-isopropylacrylamide) nanoparticles containing indocyanine green (ICG) in one pot and in water phase throughout the reaction. We have shown that copolymers of 9-decenoic acid and 1-vinylimidazole, or 9-decenoic acid alone, have an enhanced sensitivity to pH values between 7.4 and 6.8 and are superior to the widely used acrylic acid. We have also shown that incorporation of acidic comonomers leads to the favorable outcome of a higher fluorescence signal intensity in lower pH values, whereas the opposite is true of basic comonomers, where the fluorescence signal intensity is lower at low pH values. It was shown that to keep the pH response favorable the molar ratio of basic comonomers to acidic comonomers should roughly equal 1:4. We controlled the lower critical solution temperature (LCST) of the nanoparticles from around 30 to 38°C for different applications by adding acrylamide comonomers. Finally, the nanoparticles at varying pH values, when imaged by an ultrasound switchable fluorescence (USF) imaging system, showed pH sensitivity and thermosensitivity at physiological and tumor pH.

Exploring NIR Aza-BODIPY-Based Polarity Sensitive Probes with ON-and-OFF Fluorescence Switching in Pluronic Nanoparticles.

Because of their deep penetration capability in tissue, red or near infrared (NIR) fluorophores attract much attention in bio-optical imaging. Among these fluorophores, the ones that respond to the immediate microenvironment (i.e., temperature, polarity, pH, viscosity, hypoxia, etc.) are highly desirable. We studied the response of six NIR aza-BODIPY-based and structurally similar fluorophores to polarity and viscosity for incorporation inside Pluronic nanoparticles as switchable fluorescent probes (SFPs). Based on our results, all of these fluorophores were moderately to strongly sensitive to the polarity of the microenvironment. We concluded that attaching amine groups to the fluorophore is not necessary for having strong polarity sensitive probes. We further studied the response of the fluorophores when embedded inside Pluronic nanoparticles and found that four of them qualified as SFPs. We also found that the switching ratio of the fluorophore-encapsulated Pluronic nanoparticles (ION-to-IOFF) is related to the length of the hydrophobic chain of the Pluronic tri-block copolymers. As such, the highest switching ratio pertained to F-68 with the lowest hydrophobic block poly (propylene oxide) (PPO chain of only 30 units).

Hydralazine targets cAMP-dependent protein kinase leading to sirtuin1/5 activation and lifespan extension in C. elegans.

Therapeutic activation of mitochondrial function has been suggested as an effective strategy to combat aging. Hydralazine is an FDA-approved drug used in the treatment of hypertension, heart failure and cancer. Hydralazine has been recently shown to promote lifespan in C. elegans, rotifer and yeast through a mechanism which has remained elusive. Here we report cAMP-dependent protein kinase (PKA) as the direct target of hydralazine. Using in vitro and in vivo models, we demonstrate a mechanism in which binding and stabilization of a catalytic subunit of PKA by hydralazine lead to improved mitochondrial function and metabolic homeostasis via the SIRT1/SIRT5 axis, which underlies hydralazine's prolongevity and stress resistance benefits. Hydralazine also protects mitochondrial metabolism and function resulting in restoration of health and lifespan in C. elegans under high glucose and other stress conditions. Our data also provide new insights into the mechanism(s) that explain various other known beneficial effects of hydralazine.

Hydralazine induces stress resistance and extends C. elegans lifespan by activating the NRF2/SKN-1 signalling pathway.

Nuclear factor (erythroid-derived 2)-like 2 and its Caenorhabditis elegans ortholog, SKN-1, are transcription factors that have a pivotal role in the oxidative stress response, cellular homeostasis, and organismal lifespan. Similar to other defense systems, the NRF2-mediated stress response is compromised in aging and neurodegenerative diseases. Here, we report that the FDA approved drug hydralazine is a bona fide activator of the NRF2/SKN-1 signaling pathway. We demonstrate that hydralazine extends healthy lifespan (~25%) in wild type and tauopathy model C. elegans at least as effectively as other anti-aging compounds, such as curcumin and metformin. We show that hydralazine-mediated lifespan extension is SKN-1 dependent, with a mechanism most likely mimicking calorie restriction. Using both in vitro and in vivo models, we go on to demonstrate that hydralazine has neuroprotective properties against endogenous and exogenous stressors. Our data suggest that hydralazine may be a viable candidate for the treatment of age-related disorders.

Re-evaluation of biotin-streptavidin conjugation in Förster resonance energy transfer applications.

Bioaffinity conjugation between streptavidin (SA) and biotin has been widely used to link donors and acceptors for investigating the distance-dependent Förster resonance energy transfer (FRET). When studying a commonly used FRET system of (QD-SA)-(biotin-DNA-dye) [donor: quantum dot (QD); acceptor: small organic fluorescent dye; and linker: deoxyribose nucleic acid (DNA) molecule via SA-biotin conjugation], however, a contradictory finding was recently reported in the literature. It was found that the FRET lost its dependence on the number of DNA base pairs when using a phosphate-buffered saline (PBS) solution. We found that the conflicted results were caused by the ionic strength of the adopted buffer solutions. Our results suggest that the dependent FRET on the number of DNA bases is favorable in a low-ionic-strength buffer, whereas in relatively high-ionic-strength buffers, the FRET loses the DNA length dependence. We propose that the independence is mainly caused by the conformational change of DNA molecules from a stretched to a coiled mode when the cations in the high-ionic-strength buffer neutralize the negatively charged backbone of DNA molecules, thereby bringing the acceptors close to the donors.

Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity.

Giant unilamellar vesicles (GUVs) containing cholesterol often have a wide distribution in lipid composition. In this study, GUVs of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)/cholesterol and 1,2-diphytanoyl-sn-glycero-3-phosphocholine(diPhyPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesterol were prepared from dry lipid films using the standard electroformation method as well as a modified method from damp lipid films, which are made from compositional uniform liposomes prepared using the Rapid Solvent Exchange (RSE) method. We quantified the lipid compositional distributions of GUV by measuring the miscibility transition temperature of GUVs using fluorescence microscopy, since a narrower distribution in the transition temperature should correspond to a more uniform distribution in GUV lipid composition. Cholesterol molecules can demix from other lipids in dry state and form cholesterol crystals. Using optical microscopy, micron-sized crystals were observed in some dry lipid films. Thus, a major cause of GUV lipid compositional heterogeneity is the demixing of lipids in the dry film state. By avoiding the dry film state, GUVs prepared from damp lipid films have a better uniformity in lipid composition, and the standard deviations of miscibility transition temperature are about 2.5 times smaller than that of GUVs prepared from dry lipid films. Comparing the two ternary systems, diPhyPC/DPPC/cholesterol GUVs has a larger cholesterol compositional heterogeneity, which directly correlates with the low maximum solubility of cholesterol in diPhyPC lipid bilayers (40.2±0.5mol%) measured by light scattering. Our data indicate that cholesterol interacts far less favorably with diPhyPC than it does with other PCs. The damp lipid film method also has a potential of preparing GUVs from cell membranes containing native proteins without going through a dry state.