Functional RNAs: combined assembly and packaging in VLPs.

We describe here a one pot RNA production, packaging and delivery system based on bacteriophage Qß. We demonstrate a method for production of a novel RNAi scaffold, packaged within Qß virus-like particles (VLPs). The RNAi scaffold is a general utility chimera that contains a functional RNA duplex with paired silencing and carrier sequences stabilized by a miR-30 stem-loop. The Qß hairpin on the 5΄ end confers affinity for the Qß coat protein (CP). Silencing sequences can include mature miRNAs and siRNAs, and can target essentially any desired mRNA. The VLP-RNAi assembles upon co-expression of CP and the RNAi scaffold in E. coli. The annealing of the scaffold to form functional RNAs is intramolecular and is therefore robust and concentration independent. We demonstrate dose- and time-dependent inhibition of GFP expression in human cells with VLP-RNAi. In addition, we target the 3΄UTR of oncogenic Ras mRNA and suppress Pan-Ras expression, which attenuates cell proliferation and promotes mortality of brain tumor cells. This combination of RNAi scaffold design with Qß VLP packaging is demonstrated to be target-specific and efficient.

Effect of laser fluence, nanoparticle concentration and total energy input per cell on photoporation of cells.

Intracellular delivery of molecules can be increased by laser-exposure of carbon black nanoparticles to cause photoporation of the cells. Here we sought to determine effects of multiple laser exposure parameters on intracellular uptake and cell viability with the goal of determining a single unifying parameter that predicts cellular bioeffects. DU145 human prostate cancer cells in suspension with nanoparticles were exposed to near-infrared nanosecond laser pulses over a range of experimental conditions. Increased bioeffects (i.e., uptake and viability loss determined by flow cytometry) were seen when increasing laser fluence, number of pulses and nanoparticle concentration, and decreasing cell concentration. Bioeffects caused by different combinations of these four parameters were generally predicted by their cumulative energy input per cell, which served as a unifying parameter. This indicates that photoporation depends on what appears to be the cumulative effect of multiple cell-nanoparticle interactions from neighboring nanoparticles during a series of laser pulses.

Energy Transfer Mechanisms during Molecular Delivery to Cells by Laser-Activated Carbon Nanoparticles.

Previous studies have shown that exposure of carbon black nanoparticles to nanosecond pulsed near-infrared laser causes intracellular delivery of molecules through hypothesized transient breaks in the cell membrane. The goal of this study is to determine the underlying mechanisms of sequential energy transfer from laser light to nanoparticle to fluid medium to cell. We found that laser pulses on a timescale of 10 ns rapidly heat carbon nanoparticles to temperatures on the order of 1200 K. Heat is transferred from the nanoparticles to the surrounding aqueous medium on a similar timescale, causing vaporization of the surrounding water and generation of acoustic emissions. Nearby cells can be impacted thermally by the hot bubbles and mechanically by fluid mechanical forces to transiently increase cell membrane permeability. The experimental and theoretical results indicate that transfer of momentum and/or heat from the bubbles to the cells are the dominant mechanisms of energy transfer that results in intracellular uptake of molecules. We further conclude that neither thermal expansion of the nanoparticles nor a carbon-steam chemical reaction play a significant role in the observed effects on cells, and that acoustic pressure appears to be concurrent with, but not essential to, the observed bioeffects.

Eukaryotic Ribosomal Expansion Segments as Antimicrobial Targets.

Diversity in eukaryotic rRNA structure and function offers possibilities of therapeutic targets. Unlike ribosomes of prokaryotes, eukaryotic ribosomes contain species-specific rRNA expansion segments (ESs) with idiosyncratic structures and functions that are essential and specific to some organisms. Here we investigate expansion segment 7 (ES7), one of the largest and most variable expansions of the eukaryotic ribosome. We hypothesize that ES7 of the pathogenic fungi Candida albicans (ES7CA) could be a prototypic drug target. We show that isolated ES7CA folds reversibly to a native-like state. We developed a fluorescence displacement assay using an RNA binding fluorescent probe, F-neo. F-neo binds tightly to ES7CA with a Kd of 2.5 × 10-9 M but binds weakly to ES7 of humans (ES7HS) with a Kd estimated to be greater than 7 µM. The fluorescence displacement assay was used to investigate the affinities of a library of peptidic aminosugar conjugates (PAs) for ES7CA. For conjugates with highest affinities for ES7CA (NeoRH, NeoFH, and NeoYH), the lowest dose needed to induce mortality in C. albicans (minimum inhibitory concentration, MIC) was determined. PAs with the lowest MIC values were tested for cytotoxicity in HEK293T cells. Molecules with high affinity for ES7CA in vitro induce mortality in C. albicans but not in HEK293T cells. The results are consistent with the hypothesis that ESs represent useful targets for chemotherapeutics directed against eukaryotic pathogens.

Role of cytoskeletal mechanics and cell membrane fluidity in the intracellular delivery of molecules mediated by laser-activated carbon nanoparticles.

Exposure of cells and nanoparticles to near-infrared nanosecond pulsed laser light can lead to efficient intracellular delivery of molecules while maintaining high cell viability by a photoacoustic phenomenon known as transient nanoparticle energy transduction (TNET). Here, we examined the influence of cytoskeletal mechanics and plasma membrane fluidity on intracellular uptake of molecules and loss of cell viability due to TNET. We found that destabilization of actin filaments using latrunculin A led to greater uptake of molecules and less viability loss caused by TNET. Stabilization of actin filaments using jasplakinolide had no significant effect on uptake or viability loss caused by TNET. To study the role of plasma membrane fluidity, we increased fluidity by depletion of membrane cholesterol using methyl-ß-cyclodextrin and decreased fluidity by enrichment of the membrane with cholesterol using water-soluble cholesterol. Neither of these membrane fluidity changes significantly altered cellular uptake or viability loss caused by TNET. We conclude that weakening mechanical integrity of the cytoskeleton can increase intracellular uptake and decrease loss of cell viability, while plasma membrane fluidity does not appear to play a significant role in uptake or viability loss caused by TNET. The positive effects of cytoskeletal weakening may be due to an enhanced ability of the cell to recover from the effects of TNET and maintain viability. Biotechnol. Bioeng. 2017;114: 2390-2399. © 2017 Wiley Periodicals, Inc.

Photoporation Using Carbon Nanotubes for Intracellular Delivery of Molecules and Its Relationship to Photoacoustic Pressure.

Exposure of carbon-black (CB) nanoparticles to near-infrared nanosecond-pulsed laser energy can cause efficient intracellular delivery of molecules by photoporation. Here, cellular bioeffects of multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) are compared to those of CB nanoparticles. In DU145 prostate-cancer cells, photoporation using CB nanoparticles transitions from (i) cells with molecular uptake to (ii) nonviable cells to (iii) fragmented cells with increasing laser fluence, as seen previously. In contrast, photoporation with MWCNTs causes uptake and, at higher fluence, fragmentation, but does not generate nonviable cells, and SWCNTs show little evidence of bioeffects, except at extreme laser conditions, which generate nonviable cells and fragmentation, but no significant uptake. These different behaviors cannot be explained by photoacoustic pressure output from the particles. All particle types emit a single, ≈100 ns, mostly positive-pressure pulse that increases in amplitude with laser fluence. Different particle types emit different peak pressures, which are highest for SWCNTs, followed by CB nanoparticles and then MWCNTs, which does not correlate with cellular bioeffects between different particle types. This study concludes that cellular bioeffects depend strongly on the type of carbon nanoparticle used during photoporation and that photoacoustic pressure is unlikely to play a direct mechanistic role in the observed bioeffects.