Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo. Our data showed that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology is, to our knowledge, the first noninvasive approach for capturing mRNA from live single cells in their natural microenvironment.

Caging metal ions with visible light-responsive nanopolymersomes.

Polymersomes are bilayer vesicles that self-assemble from amphiphilic diblock copolymers, and provide an attractive system for the delivery of biological and nonbiological molecules due to their environmental compatibility, mechanical stability, synthetic tunability, large aqueous core, and hyperthick hydrophobic membrane. Herein, we report a nanoscale photoresponsive polymersome system featuring a meso-to-meso ethyne-bridged bis[(porphinato)zinc] (PZn2) fluorophore hydrophobic membrane solute and dextran in the aqueous core. Upon 488 nm irradiation in solution or in microinjected zebrafish embryos, the polymersomes underwent deformation, as monitored by a characteristic red-shifted PZn2 emission spectrum and confirmed by cryo-TEM. The versatility of this system was demonstrated through the encapsulation and photorelease of a fluorophore (FITC), as well as two different metal ions, Zn(2+) and Ca(2+).

Caged oligonucleotides for bidirectional photomodulation of let-7 miRNA in zebrafish embryos.

Many biological functions of microRNA (miRNA) have been identified in the past decade. However, a single miRNA can regulate multiple gene targets, thus it has been a challenge to elucidate the specific functions of each miRNA in different locations and times. New chemical tools make it possible to modulate miRNA activity with higher spatiotemporal resolution. Here, we describe light-activated (caged) constructs for switching let-7 miRNA 'on' or 'off' with 365 nm light in developing zebrafish embryos.

Polymersome Poration and Rupture Mediated by Plasmonic Nanoparticles in Response to Single-Pulse Irradiation.

The self-assembly of amphiphilic diblock copolymers into polymeric vesicles, commonly known as polymersomes, results in a versatile system for a variety of applications including drug delivery and microreactors. In this study, we show that the incorporation of hydrophobic plasmonic nanoparticles within the polymersome membrane facilitates light-stimulated release of vesicle encapsulants. This work seeks to achieve tunable, triggered release with non-invasive, spatiotemporal control using single-pulse irradiation. Gold nanoparticles (AuNPs) are incorporated as photosensitizers into the hydrophobic membrane of micron-scale polymersomes and the cargo release profile is controlled by varying the pulse energy and nanoparticle concentration. We have demonstrated the ability to achieve immediate vesicle rupture as well as vesicle poration resulting in temporal cargo diffusion. Additionally, changing the pulse duration, from femtosecond to nanosecond, provides mechanistic insight into the photothermal and photomechanical contributors that govern membrane disruption in this polymer-nanoparticle hybrid system.

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.