Near-Infrared Induced miR-34a Delivery from Nanoparticles in Esophageal Cancer Treatment.

Current nucleic acid delivery methods have not achieved efficient, non-toxic delivery of miRNAs with tumor-specific selectivity. In this study, a new delivery system based on light-inducible gold-silver-gold, core-shell-shell (CSS) nanoparticles is presented. This system delivers small nucleic acid therapeutics with precise spatiotemporal control, demonstrating the potential for achieving tumor-specific selectivity and efficient delivery of miRNA mimics. The light-inducible particles leverage the photothermal heating of metal nanoparticles due to the local surface plasmonic resonance for controlled chemical cleavage and release of the miRNA mimic payload. The CSS morphology and composition result in a plasmonic resonance within the near-infrared (NIR) region of the light spectrum. Through this method, exogenous miR-34a-5p mimics are effectively delivered to human squamous cell carcinoma TE10 cells, leading to apoptosis induction without adverse effects on untransformed keratinocytes in vitro. The CSS nanoparticle delivery system is tested in vivo in Foxn1nu athymic nude mice with bilateral human esophageal TE10 cancer cells xenografts. These experiments reveal that this CSS nanoparticle conjugates, when systemically administered, followed by 850 nm light emitting diode irradiation at the tumor site, 6 h post-injection, produce a significant and sustained reduction in tumor volume, exceeding 87% in less than 72 h.

Delivery of Therapeutic miR-148b Mimic via Poly(ß Amino Ester) Polyplexes for Post-transcriptional Gene Regulation and Apoptosis of A549 Cells.

In this study, we utilized selectively modified, biodegradable polymer-based polyplexes to deliver custom, exogenous miR-148b mimics to induce apoptosis in human lung cancer (A549) cells. The gene regulatory effects of the payload miRNA mimics (miR-148b-3p) were first evaluated through bioinformatic analyses to uncover specific gene targets involved in critical carcinogenic pathways. Hyperbranched poly(ß amino ester) polyplexes (hPBAE) loaded with custom miR-148b mimics were then developed for targeted therapy. When evaluated in vitro, these hPBAE-based polyplexes sustained high intracellular uptake, low cytotoxicity, and efficient escape from endosomes to deliver functionally intact miRNA mimics to the cytosol. High-resolution confocal microscopy revealed successful intracellular uptake, cell viability was assessed through qualitative fluorescence microscopy and fluorescence-based DNA quantification, and successful cytosolic delivery of intact miRNA mimics was evaluated using real-time polymerase chain reaction (RT-PCR) to demonstrate target gene knockdown. The hPBAE-miRNA mimic polyplexes were shown to induce apoptosis among A549 cells through direct modulation of intracellular protein expression, targeting multiple potential carcinogenic pathways at the gene level. These results indicated that spatially controlled miR-148b mimic delivery can promote efficient cancer cell death in vitro and may lead to an enhanced therapeutic design for in vivo application.

Liquid-Cell Electron Tomography of Biological Systems.

Liquid-cell electron microscopy is a rapidly growing field in the imaging domain. While real-time observations are readily available to analyze materials and biological systems, these measurementshave been limited to the two-dimensional (2-D) image plane. Here, we introduce an exciting technical advance to image materials in 3-D while enclosed in liquid. The development of liquid-cell electron tomography permitted us to observe and quantify host-pathogen interactions in solution while contained in the vacuum system of the electron microscope. In doing so, we demonstrate new insights for the rules of engagement involving a unique bacteriophage and its host bacterium. A deeper analysis of the genetic content of the phage pathogens revealed structural features of the infectious units while introducing a new paradigm for host interactions. Overall, we demonstrate a technological opportunity to elevate research efforts for in situ imaging while providing a new level of dimensionality beyond the current state of the field.

Cryo-EM-On-a-Chip: Custom-Designed Substrates for the 3D Analysis of Macromolecules.

The fight against human disease requires a multidisciplinary scientific approach. Applying tools from seemingly unrelated areas, such as materials science and molecular biology, researchers can overcome long-standing challenges to improve knowledge of molecular pathologies. Here, custom-designed substrates composed of silicon nitride (SiN) are used to study the 3D attributes of tumor suppressor proteins that function in DNA repair events. New on-chip preparation strategies enable the isolation of native protein complexes from human cancer cells. Combined techniques of cryo-electron microscopy (EM) and molecular modeling reveal a new modified form of the p53 tumor suppressor present in aggressive glioblastoma multiforme cancer cells. Taken together, the findings provide a radical new design for cryo-EM substrates to evaluate the structures of disease-related macromolecules.

Correcting errors in the BRCA1 warning system.

Given its important role in human health and disease, remarkably little is known about the full-length three-dimensional (3D) molecular architecture of the breast cancer type 1 susceptibility protein (BRCA1), or its mechanisms to engage the tumor suppressor, TP53 (p53). Here, we show how a prevalent cancer-related mutation in the C-terminal region of the full-length protein, BRCA15382insC, affects its structural properties, yet can be biochemically corrected to restore its functional capacity. As a downstream consequence of restoring the ubiquitin ligase activity of mutated BRCA15382insC, the DNA repair response of p53 was enhanced in cellular extracts naturally deficient in BRCA1 protein expression. Complementary structural insights of p53 tetramers bound to DNA in different stage of the repair process support these biochemical findings in the context of human cancer cells. Equally important, we show how this knowledge can be used to lower the viability of breast cancer cells by modulating the stability of the BRCA1 protein and its associated players.

Structural analysis of BRCA1 reveals modification hotspot.

Cancer cells afflicted with mutations in the breast cancer susceptibility protein (BRCA1) often suffer from increased DNA damage and genomic instability. The precise manner in which physical changes to BRCA1 influence its role in DNA maintenance remains unclear. We used single-particle electron microscopy to study the three-dimensional properties of BRCA1 naturally produced in breast cancer cells. Structural studies revealed new information for full-length BRCA1, engaging its nuclear binding partner, the BRCA1-associated RING domain protein (BARD1). Equally important, we identified a region in mutated BRCA1 that was highly susceptible to ubiquitination. We refer to this site as a modification "hotspot." Ubiquitin adducts in the hotspot region proved to be biochemically reversible. Collectively, we show how key changes to BRCA1 affect its structure-function relationship, and present new insights to potentially modulate mutated BRCA1 in human cancer cells.