Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

Glioblastoma Multiforme (GBM) is a highly prevalent and deadly brain malignancy characterized by poor prognosis and restricted disease management potential. Despite the success of nanocarrier systems to improve drug/gene therapy for cancer, active targeting specificity remains a major hurdle for GBM. Additionally, since the brain is a multi-cell type organ, there is a critical need to develop an approach to distinguish between GBM cells and healthy brain cells for safe and successful treatment. In this report, we have incorporated hyaluronic acid (HA) as an active targeting ligand for GBM. To do so, we employed HA conjugated liposomes (HALNPs) to study the uptake pathway in key cells in the brain including primary astrocytes, microglia, and human GBM cells. We observed that the HALNPs specifically target GBM cells over other brain cells due to higher expression of CD44 in tumor cells. Furthermore, CD44 driven HALNP uptake into GBM cells resulted in lysosomal evasion and increased efficacy of Doxorubicin, a model anti-neoplastic agent, while the astrocytes and microglia cells exhibited extensive HALNP-lysosome co-localization and decreased antineoplastic potency. In summary, novel CD44 targeted lipid based nanocarriers appear to be proficient in mediating site-specific delivery of drugs via CD44 receptors in GBM cells, with an improved therapeutic margin and safety.

Titanium dioxide (TiO2) nanoparticles are one of the most highly manufactured and employed nanomaterials in the world with applications in copious industrial and consumer products. The liver is a major accumulation site for many nanoparticles, including TiO2, directly through intentional exposure or indirectly through unintentional ingestion via water, food or animals and increased environmental contamination. Growing concerns over the current usage of TiO2 coupled with the lack of mechanistic understanding of its potential health risk is the motivation for this study. Here we determined the toxic effect of three different TiO2 nanoparticles (commercially available rutile, anatase and P25) on primary rat hepatocytes. Specifically, we evaluated events related to hepatocyte functions and mitochondrial dynamics: (1) urea and albumin synthesis using colorimetric and ELISA assays, respectively; (2) redox signaling mechanisms by measuring reactive oxygen species (ROS) production, manganese superoxide dismutase (MnSOD) activity and mitochondrial membrane potential (MMP); (3) OPA1 and Mfn-1 expression that mediates the mitochondrial dynamics by PCR; and (4) mitochondrial morphology by MitoTracker Green FM staining. All three TiO2 nanoparticles induced a significant loss (p < 0.05) in hepatocyte functions even at concentrations as low as 50 ppm with commercially used P25 causing maximum damage. TiO2 nanoparticles induced a strong oxidative stress in primary hepatocytes. TiO2 nanoparticles exposure also resulted in morphological changes in mitochondria and substantial loss in the fusion process, thus impairing the mitochondrial dynamics. Although this study demonstrated that TiO2 nanoparticles exposure resulted in substantial damage to primary hepatocytes, more in vitro and in vivo studies are required to determine the complete toxicological mechanism in primary hepatocytes and subsequently liver function.

Mechanical properties of the cellular microenvironment induces astrogliosisin vitroin primary rat astrocytes.