A Facile NMR Method for Pre-MRI Evaluation of Trigger-Responsive T1 Contrast Enhancement.

There is a growing interest in developing paramagnetic nanoparticles as responsive magnetic resonance imaging (MRI) contrast agents, which feature switchable T1 image contrast of water protons upon biochemical cues for better discerning diseases. However, performing an MRI is pragmatically limited by its cost and availability. Hence, a facile, routine method for measuring the T1 contrast is highly desired in early-stage development. This work presents a single-point inversion recovery (IR) nuclear magnetic resonance (NMR) method that can rapidly evaluate T1 contrast change by employing a single, optimized IR pulse sequence that minimizes water signal for "off-state" nanoparticles and allows for sensitively measuring the signal change with "switch-on" T1 contrast. Using peptide-induced liposomal gadopentetic acid (Gd3+ -DTPA) release and redox-sensitive manganese oxide (MnO2 ) nanoparticles as a demonstration of generality, this method successfully evaluates the T1 shortening of water protons caused by liposomal Gd3+ -DTPA release and Mn2+ formation from MnO2 reduction. Furthermore, the NMR measurement is highly correlated to T1 -weighted MRI scans, suggesting its feasibility to predict the MRI results at the same field strength. This NMR method can be a low-cost, time-saving alternative for pre-MRI evaluation for a diversity of responsive T1 contrast systems.

Constructing pH-Responsive and Tunable Azo-Phenol-Based o-Nitrobenzyl Photocages.

Multiple triggered-release strategies are widely utilized to control the release of caged target molecules. Among them, photocages with conditional triggers provide extra layers of control in photorelease. In this work, a series of pH-responsive photocages was designed that could be triggered under irradiation and specific intracellular pH values. pH-sensitive phenolic groups were conjugated with o-nitrobenzyl (oNB) to form azo-phenolic NPX photocages with tunable pKa. These azo-phenol-based oNB photocages showed differentiable photoreleasing profiles at pH 5.0, 7.2 and 9.0. By attaching fluorogenic cargos, it was shown that one of the photocages, NPdiCl, could be used to differentiate between acidic pH 5.0 and neutral pH 7.2 in cells under artificial pH conditions. Finally, NPdiCl was identified as a promising pH-responsive photocage for photoreleasing cargo inside acidic tumor cells.

Nano-On-Nano: Responsive Nanosubstrate-Mediated Liposome Delivery with High Cellular Uptake Efficiency.

Efficiently delivering liposomal content to cells in a relatively uniform dose and patterned fashion, especially bypassing the degradative endocytosis pathway, is an important technology in cell culture and potentially to tissue engineering that still remains challenging. We developed a "nano-on-nano" platform technology that consists of the following three material features: (1) high density silicon nanopillars to create a pseudo-3-dimensional nanoenvironment for cell culturing, (2) thermoresponsive polymer grafted onto silicon nanopillars to form a responsive nanosubstrate, and (3) immobilized liposomes using a biotin-streptavidin-biotin conjugation. The working principle is that the liposomes are detached for cellular uptake upon thermal stimulation and high local liposome concentration between the cells and substrates drives the cellular uptake with nonendocytic pathways. Cryo-EM images confirms that liposomes are attached to form liposome-warped nanopillars. Upon thermal stimulation, an 8 times higher increase in the liposomal fluorescence intensity is observed compared to the conventional solution-phase liposome delivery, indicating that high local concentration drives liposome uptake with greater efficiency. Moreover, preliminary mechanistic studies reveal that these liposomes are taken up by nonendocytic pathways. The ability of our nano-on-nano delivery system that achieves efficient dose-uniform cellular delivery can open a unique era in cell and tissue engineering by controlling cell behaviors with the delivery of bioactive ingredient-loaded liposomes.

A Toolkit for Engineering Proteins in Living Cells: Peptide with a Tryptophan-Selective Ru-TAP Complex to Regioselectively Photolabel Specific Proteins.

Using a chemical approach to crosslink functionally versatile bioeffectors (such as peptides) to native proteins of interest (POI) directly inside a living cell is a useful toolbox for chemical biologists. However, this goal has not been reached due to unsatisfactory chemoselectivity, regioselectivity, and protein selectivity in protein labeling within living cells. Herein, we report the proof of concept of a cytocompatible and highly selective photolabeling strategy using a tryptophan-specific Ru-TAP complex as a photocrosslinker. Aside from the high selectivity, the photolabeling is blue light-driven by a photoinduced electron transfer (PeT) and allows the bioeffector to bear an additional UV-responsive unit. The two different photosensitivities are demonstrated by blue light-photocrosslinking a UV-sensitive peptide to POI. Our visible light photolabeling can generate photocaged proteins for subsequent activity manipulation by UV light. Cytoskeletal dynamics regulation is demonstrated in living cells via the unprecedented POI photomanipulation and proves that our methodology opens a new avenue to endogenous protein modification.

Liposomal PHD2 Inhibitors and the Enhanced Efficacy in Stabilizing HIF-1α.

Prolyl hydroxylase domain-containing protein 2 (PHD2) inhibition, which stabilizes hypoxia-inducible factor (HIF)-1α and thus triggers adaptation responses to hypoxia in cells, has become an important therapeutic target. Despite the proven high potency, small-molecule PHD2 inhibitors such as IOX2 may require a nanoformulation for favorable biodistribution to reduce off-target toxicity. A liposome formulation for improving the pharmacokinetics of an encapsulated drug while allowing a targeted delivery is a viable option. This study aimed to develop an efficient loading method that can encapsulate IOX2 and other PHD2 inhibitors with similar pharmacophore features in nanosized liposomes. Driven by a transmembrane calcium acetate gradient, a nearly 100% remote loading efficiency of IOX2 into liposomes was achieved with an optimized extraliposomal solution. The electron microscopy imaging revealed that IOX2 formed nanoprecipitates inside the liposome's interior compartments after loading. For drug efficacy, liposomal IOX2 outperformed the free drug in inducing the HIF-1α levels in cell experiments, especially when using a targeting ligand. This method also enabled two clinically used inhibitors-vadadustat and roxadustat-to be loaded into liposomes with a high encapsulation efficiency, indicating its generality to load other heterocyclic glycinamide PHD2 inhibitors. We believe that the liposome formulation of PHD2 inhibitors, particularly in conjunction with active targeting, would have therapeutic potential for treating more specifically localized disease lesions.

Immune cell shuttle for precise delivery of nanotherapeutics for heart disease and cancer.

The delivery of therapeutics through the circulatory system is one of the least arduous and less invasive interventions; however, this approach is hampered by low vascular density or permeability. In this study, by exploiting the ability of monocytes to actively penetrate into diseased sites, we designed aptamer-based lipid nanovectors that actively bind onto the surface of monocytes and are released upon reaching the diseased sites. Our method was thoroughly assessed through treating two of the top causes of death in the world, cardiac ischemia-reperfusion injury and pancreatic ductal adenocarcinoma with or without liver metastasis, and showed a significant increase in survival and healing with no toxicity to the liver and kidneys in either case, indicating the success and ubiquity of our platform. We believe that this system provides a new therapeutic method, which can potentially be adapted to treat a myriad of diseases that involve monocyte recruitment in their pathophysiology.

Near-infrared fluorescent probes based on TBET and FRET rhodamine acceptors with different pK a values for sensitive ratiometric visualization of pH changes in live cells.

Three near-infrared ratiometric fluorescent probes (A-C) based on TBET and FRET near-infrared rhodamine acceptors with different pK a values were designed and synthesized to achieve sensitive ratiometric visualization of pH variations in lysosomes in visible and near-infrared channels. Tetraphenylethene (TPE) was bonded to near-infrared rhodamine dyes through short electrical π -conjugation linkers to prevent an aggregation-caused quenching (ACQ) effect and allow highly efficient energy transfer of up to 98.9% from TPE donors to rhodamine acceptors. Probes A-C respond to pH variation from 7.4 to 3.0 in both buffer solutions and live cells with significant decreases of donor fluorescence and concomitant extraordinary increases of rhodamine acceptor fluorescence because of highly efficient energy transfer. In addition, probe C is capable of determining pH fluctuations in live cells treated with chloroquine. The probes show good photostability, excellent cell membrane permeability, high selectivity to pH, and two well-resolved emission peaks to ensure accurately comparative and quantitative analyses of intracellular pH changes.

Lipid-Wrapped Upconversion Nanoconstruct/Photosensitizer Complex for Near-Infrared Light-Mediated Photodynamic Therapy.

Photodynamic therapy (PDT) is a noninvasive medical technology that has been applied in cancer treatment where it is accessible by direct or endoscope-assisted light irradiation. To lower phototoxicity and increase tissue penetration depth of light, great effort has been focused on developing new sensitizers that can utilize red or near-infrared (NIR) light for the past decades. Lanthanide-doped upconversion nanoparticles (UCNPs) have a unique property to transduce NIR excitation light to UV-vis emission efficiently. This property allows some low-cost, low-toxicity, commercially available visible light sensitizers, which originally are not suitable for deep tissue PDT, to be activated by NIR light and have been reported extensively in the past few years. However, some issues still remain in the UCNP-assisted PDT platform such as colloidal stability, photosensitizer loading efficiency, and accessibility for targeting ligand installation, despite some advances in this direction. In this study, we designed a facile phospholipid-coated UCNP method to generate a highly colloidally stable nanoplatform that can effectively load a series of visible light sensitizers in the lipid layers. The loading stability and singlet oxygen generation efficiency of this sensitizer-loaded lipid-coated UCNP platform were investigated. We also have demonstrated the enhanced cellular uptake efficiency and tumor cell selectivity of this lipid-coated UCNP platform by changing the lipid dopant. On the basis of the evidence of our results, the lipid-complexed UCNP nanoparticles could serve as an effective photosensitizer carrier for NIR light-mediated PDT.

A Highly-Efficient Single Segment White Random Laser.

Production of multicolor or multiple wavelength lasers over the full visible-color spectrum from a single chip device has widespread applications, such as superbright solid-state lighting, color laser displays, light-based version of Wi-Fi (Li-Fi), and bioimaging, etc. However, designing such lasing devices remains a challenging issue owing to the material requirements for producing multicolor emissions and sophisticated design for producing laser action. Here we demonstrate a simple design and highly efficient single segment white random laser based on solution-processed NaYF4:Yb/Er/Tm@NaYF4:Eu core-shell nanoparticles assisted by Au/MoO3 multilayer hyperbolic meta-materials. The multicolor lasing emitted from core-shell nanoparticles covering the red, green, and blue, simultaneously, can be greatly enhanced by the high photonic density of states with a suitable design of hyperbolic meta-materials, which enables decreasing the energy consumption of photon propagation. As a result, the energy upconversion emission is enhanced by ∼50 times with a drastic reduction of the lasing threshold. The multiple scatterings arising from the inherent nature of the disordered nanoparticle matrix provide a convenient way for the formation of closed feedback loops, which is beneficial for the coherent laser action. The experimental results were supported by the electromagnetic simulations derived from the finite-difference time-domain (FDTD) method. The approach shown here can greatly simplify the design of laser structures with color-tunable emissions, which can be extended to many other material systems. Together with the characteristics of angle free laser action, our device provides a promising solution toward the realization of many laser-based practical applications.

New Near-infrared Fluorescent Probes with Single-photon Anti-Stokes-shift Fluorescence for Sensitive Determination of pH Variances in Lysosomes with a Double-Checked Capability.

Two near-infrared luminescent probes with Stokes-shift and single-photon anti-Stokes-shift fluorescence properties for sensitive determination of pH variance in lysosomes have been synthesized. A morpholine residue in probe A which serves as a targeting group for lysosomes in viable cells was attached to the fluorophores via a spirolactam moiety while a mannose residue was ligated to probe B resulting in increased biocompatibility and solubility in water. Probes A and B contain closed spirolactam moieties, and show no Stokes-shift or anti-Stokes-shift fluorescence under neutral or alkali conditions. However, the probes incrementally react to pH variance from 7.22 to 2.76 with measurable increases in both Stokes-shift and anti-Stokes-shift fluorescence at 699 nm and 693 nm under 645 nm and 800 nm excitation, respectively. This acid-activated fluorescence is produced by the breaking of the probe spirolactam moiety, which greatly increased overall π-conjugation in the probes. These probes possess upconversion near-infrared fluorescence imaging advantages including minimum cellular photo-damage, tissue penetration, and minimum biological fluorescence background. They display excellent photostability with low dye photobleaching and show good biocompatibility. They are selective and capable of detecting pH variances in lysosomes at excitation with two different wavelengths, i.e., 645 and 800 nm.

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

Luminescent Probes for Sensitive Detection of pH Changes in Live Cells through Two Near-Infrared Luminescence Channels.

Two water-soluble near-infrared luminescent probes, which possess both conventional intense Stokes fluorescence and unique single-photon frequency upconversion luminescence (FUCL), were developed for sensitive and selective detection of pH changes in live cells. The water solubility and biocompatibility of these probes were achieved by introducing mannose residues through 2,2'-(ethylenedioxy)diethylamine tethered spacers to a near-infrared conventional fluorescence (CF) and FUCL organic fluorophore. At a pH higher than 7.4, the probes have ring-closed spirocyclic lactam structures, thus are colorless and nonfluorescent. Nevertheless, they sensitively respond to acidic pH values, with a drastic structural change to ring-opened spirocyclic lactam forms, which cause significant absorbance increases at 714 nm. Correspondingly, their near-infrared CF and FUCL intensities at 740 nm are also significantly enhanced when excited by 690 and 808 nm, respectively. The probes hold a variety of advantages such as high sensitivity, excellent reversibility and selectivity to pH over metal ions, low cellular autofluorescence background interference, good cell membrane permeability and photostability, as well as low cytotoxicity. Our results have successfully proven that these probes can visualize intracellular lysosomal pH changes in live cells by monitoring both near-infrared CF and FUCL changes.

Efficacy of Mastoparan-AF alone and in combination with clinically used antibiotics on nosocomial multidrug-resistant Acinetobacter baumannii.

Emergence of multidrug-resistant Acinetobacter baumannii (MDRAB) has become a critical clinical problem worldwide and limited therapeutic options for infectious diseases caused by MDRAB. Therefore, there is an urgent need for the development of new antimicrobial agents or alternative therapy to combat MDRAB infection. The aim of this study was to investigate effects of Mastoparan-AF (MP-AF), an amphipathic peptide isolated from the hornet venom of Vespa affinis with broad-spectrum antimicrobial activity, on MDRAB. As compared with clinical used antibiotics, MP-AF exhibited potent antimicrobial activity at 2-16 µg/ml against the reference strain A. baumannii ATCC 15151 and seven MDRAB clinical isolates, especially the colistin-resistant MDRAB, E0158. The synergistic antimicrobial combination study revealed that MP-AF acted synergistically with specific antibiotics, e.g., ciprofloxacin, trimethoprim/sulfamethoxazole (SXT) or colistin against some isolates of the MDRAB. It was noteworthy when MP-AF combined with SXT exhibited synergistic activity against all SXT-resistant MDRAB isolates. The synergistic combination of MP-AF and antibiotics could reduce the dosage recommended of each antimicrobial agent and improve the safety of medications with ignorable adverse effects, such as colistin with nephrotoxicity in therapeutic dose. Furthermore, MP-AF combined with antibiotics with different antimicrobial mechanisms could reduce selective pressure of antibiotics on bacteria and prevent the emergence of antimicrobial-resistant strains. Importantly, we are the first finding that MP-AF could make MDRAB from the original non-susceptibility to SXT become sensitivity. In conclusion, MP-AF alone or in combination with other antibiotics, especially SXT, is a potential candidate against MDRAB infection in clinical medicine.

Targeted and efficient activation of channelrhodopsins expressed in living cells via specifically-bound upconversion nanoparticles.

Optogenetics is an innovative technology now widely adopted by researchers in different fields of biological sciences. However, most light-sensitive proteins adopted in optogenetics are excited by ultraviolet or visible light which has a weak tissue penetration capability. Upconversion nanoparticles (UCNPs), which absorb near-infrared (NIR) light to emit shorter wavelength light, can help address this issue. In this report, we demonstrated the target selectivity by specifically conjugating the UCNPs with channelrhodopsin-2 (ChR2). We tagged the V5 epitope to the extracellular N-terminal of ChR2 (V5-ChR2m) and functionalized the surface of UCNPs with NeutrAvidin (NAv-UCNPs). After the binding of the biotinylated antibody against V5 onto the V5-ChR2m expressed in the plasma membrane of live HEK293T cells, our results showed that the NAv-UCNPs were specifically bound to the membrane of cells expressing V5-ChR2m. Without the V5 epitope or NAv modification, no binding of UCNPs onto the cell membrane was observed. For the cells expressing V5-ChR2m and bound with NAv-UCNPs, both 488 nm illumination and the upconverted blue emission from UCNPs by 980 nm excitation induced an inward current and elevated the intracellular Ca2+ concentration. Our design reduces the distance between UCNPs and light-sensitive proteins to the molecular level, which not only minimizes the NIR energy required but also provides a way to guide the specific binding for optogenetics applications.

Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking.

The abnormal assembly of misfolded proteins into neurotoxic aggregates is the hallmark associated with neurodegenerative diseases. Herein, we establish a photocontrollable platform to trigger amyloidogenesis to recapitulate the pathogenesis of amyotrophic lateral sclerosis (ALS) by applying a chemically engineered probe as a "switch" in live cells. This probe is composed of an amyloidogenic peptide from TDP-43, a photolabile linker, a polycationic sequence both to mask amyloidogenicity and for cell penetration, and a fluorophore for visualization. The photocontrollable probe can self-assemble into a spherical vesicle but rapidly develops massive nanofibrils with amyloid properties upon photoactivation. The photoinduced in vitro fibrillization process is characterized by biophysical techniques. In cellular experiments, this cell-penetrable vesicle was retained in the cytoplasm, seeded the mislocalized endogenous TDP-43 into aggregates upon irradiation, and consequently initiated apoptosis. In addition, this photocontrollable vesicle interfered with nucleocytoplasmic protein transport and triggered cortical neuron degeneration. Our developed strategy provides in vitro and in vivo spatiotemporal control of neurotoxic fibrillar aggregate formation, which can be readily applied in the studies of protein misfolding, aggregation-induced protein mislocalization, and amyloid-induced pathogenesis in different diseases.

Characterization of genes encoding proteins containing HD-related output domain in Xanthomonas campestris pv. campestris.

The Gram-negative plant pathogen Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. The production of Xcc virulence factors is regulated by Clp and RpfF. HD-related output domain (HDOD) is a protein domain of unknown biochemical function. The genome of Xcc encodes three proteins (GsmR, HdpA, and HdpB) with an HDOD. The GsmR has been reported to play a role in the general stress response and cell motility and its expression is positively regulated by Clp. Here, the function and transcription of hdpA and hdpB were characterized. Mutation of hdpA resulted in enhanced bacterial attachment. In addition, the expression of hdpA was positively regulated by RpfF but not by Clp, subject to catabolite repression and affected by several stress conditions. However, mutational analysis and reporter assay showed that hdpB had no effect on the production of a range of virulence factors and its expression was independent of Clp and RpfF. The results shown here not only extend the previous work on RpfF regulation to show that it influences the expression of hdpA in Xcc, but also expand knowledge of the function of the HDOD containing proteins in bacteria.

Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle.

Photoactivatable (caged) bioeffectors provide a way to remotely trigger or disable biochemical pathways in living organisms at a desired time and location with a pulse of light (uncaging), but the phototoxicity of ultraviolet (UV) often limits its application. In this study, we have demonstrated the near-infrared (NIR) photoactivatable enzyme platform using protein kinase A (PKA), an important enzyme in cell biology. We successfully photoactivated PKA using NIR to phosphorylate its substrate, and this induced a downstream cellular response in living cells with high spatiotemporal resolution. In addition, this system allows NIR to selectively activate the caged enzyme immobilized on the nanoparticle surface without activating other caged proteins in the cytosol. This NIR-responsive enzyme-nanoparticle system provides an innovative approach to remote-control proteins and enzymes, which can be used by researchers who need to avoid direct UV irradiation or use UV as a secondary channel to turn on a bioeffector.