Design of a Biocompatible Hydroxyapatite-Based Nanovehicle for Efficient Delivery of Small Interference Ribonucleic Acid into Mouse Embryonic Stem Cells.

The small interference RNA (siRNA)-assisted RNA interference approach in stem cells for differentiating into cell-specific lineages is gaining importance for its therapeutic potential. An effective gene delivery platform is crucial to achieve this goal. In this context, self-fluorescent, cell-penetrating peptide (CPP)-functionalized hydroxyapatite nanoparticles (R8HNPs) were synthesized by a modified sol gel technique. R8HNPs were crystalline, displayed characteristic bands, and exhibited broad emission spectra from 350 to 750 nm corresponding to green and red fluorescence. The biocompatible R8HNPs displayed robust binding with siRNA and excellent uptake in R1 ESCs. This was attributed to functionalization with CPP. Moreover, the R8HNP-complexed siRNA exhibited excellent serum and room temperature stability. The NPs protected the siRNA from sonication, pH, and temperature-induced stress and efficiently delivered siRNA to trigger 80% silencing of a pluripotency marker gene, Oct4, in R1 ESCs at 48 h. The transient downregulation was also observed at the protein level. Our findings demonstrate R8HNPs as a promising delivery agent for siRNA therapeutics with the potential for lineage-specific differentiation and future applications in regenerative medicine.

Cell-free chromatin particles released from dying host cells are global instigators of endotoxin sepsis in mice.

We have earlier reported that cell-free chromatin (cfCh) particles that are released from dying cells, or those that circulate blood, can readily enter into healthy cells, illegitimately integrate into their genomes and induce dsDNA breaks, apoptosis and intense activation of inflammatory cytokines. We hypothesized that sepsis is caused by cfCh released from dying host cells following microbial infection leading to bystander host cell apoptosis and inflammation which are perpetuated in a vicious cycle with release of more cfCh from dying host cells. To test this hypothesis we used three cfCh inactivating agents namely 1) anti-histone antibody complexed nanoparticles which inactivate cfCh by binding to histones; 2) DNase I which inactivates cfCh by degrading its DNA component, and 3) a novel pro-oxidant combination of Resveratrol and Copper which, like DNase I, inactivates cfCh by degrading its DNA component. Female C57 BL/6 mice, 6-8 weeks old, were administered a single i.p. injection of LPS at a dose of 10 mg/Kg or 20 mg/Kg with or without concurrent treatment with the above cfCh inactivating agents. Administration of cfCh inactivating agents concurrently with LPS resulted in prevention of following pathological parameters: 1) release of cfCh in extra-cellular spaces of brain, lung and heart and in circulation; 2) release of inflammatory cytokines in circulation; 3) activation of DNA damage, apoptosis and inflammation in cells of thymus, spleen and in PBMCs; 4) DNA damage, apoptosis and inflammation in cells of lung, liver, heart, brain, kidney and small intestine; 5) liver and kidney dysfunction and elevation of serum lactate; 6) coagulopathy, fibrinolysis and thrombocytopenia; 7) lethality. We conclude that cfCh that are released from dying host cells in response to bacterial endotoxin represents a global instigator of sepsis. cfCh inactivation may provide a novel approach to management of sepsis in humans.