siRNA into osteoclasts for targeted manipulation of osteoclast functions in vitro and in vivo

We have recently demonstrated that C3bot1E174Q selectively delivers proteins and enzymes into cultured macrophages including primary human macrophages derived from monocytes from blood donors. Because C3-based transporters target monocytes/macrophages in general, they would not serve for a selective drug delivery into osteoclasts after a systemic application. However, a targeted local application of either wildtype C3 for Rho-inhibition in osteoclasts or C3-derived transport systems for targeted drug delivery into osteoclasts might be an appropriate approach to manipulate osteoclastogenesis and/or osteoclast functions, e.g. to improve the osseous integration of orthopaedic implants by suppressing osteoclast activity at the implant surface. Local application in bone and controlled release of C3 proteins or C3transporters from orthopaedic implant surfaces could be achieved by the use of biocompatible carriers such as resorbable polymers or hydrogels. Matrix stiffness is an important regulator of cell behavior. Stiffness has been shown to affect cell morphology and spreading, proliferation, migration, apoptosis rate, and differentiation. However, most cell studies are performed on tissue culture plastic, which largely fails to replicate the mechanics and microenvironment that cells experience in vivo. Tissue culture plastic is commonly cited as having an elastic modulus of approximately 1 GPa, whereas tissues in the body are less than 100 kPa, with brain having an elasticity less than 1 kPa, muscle around 10 kPa, and bone around 100 kPa. The effects of matrix stiffness are typically evaluated by analyzing cell behavior in different gel systems. Stiffness or elasticity can be varied by simply changing the crosslinking Rapamycin density. Several different hydrogel systems have been investigated including polyacrylamide gels, alginate, collagen, matrigel, chitosan, and hyaluronic acid. Because BMS-907351 c-Met inhibitor substrate stiffness regulates so many cellular functions, we wanted to investigate its role in the uptake of cell-penetrating peptides. Although the exact mechanism of cell-penetrating peptide uptake is still debated, investigators generally agree that uptake occurs via one or more of the endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, or through membrane destabilization or formation of transient pores. Our lab has designed and reported on a family of peptide inhibitors of mitogen kinase activated protein kinase- activated protein kinase 2, a kinase important in regulating inflammation through the regulation of proinflammatory cytokines. These inhibitors consist of a cell-penetrating peptide domain for intracellular delivery and a therapeutic domain that inhibits MK2. Recently, we demonstrated that the peptide variant YARAAARQARAKALARQLGVAA was taken up primarily through caveolae-mediated endocytosis in mesothelial cells. However, when comparing between data obtained from in vitro cell and in vivo animal models, we observed an unusual effect: concentrations of the YARA MK2 inhibitor peptide required for efficacy in cells ranged from 1000�C3000 mM; however, the concentration required for efficacy in animal models was ten to one hundred-fold less, in the range of 10�C100 mM. This phenomenon opposes what is normally observed in the pharmaceutical industry, as drug concentrations must usually increase to demonstrate efficacy when moving from cell culture to animal models due to metabolism and non-uniform distribution within the body. We hypothesized that the discrepancy observed in peptide concentration required to achieve efficacy in studies in vitro as compared to studies in vivo was due to the unrealistic stiffness of tissue culture polystyrene. Using a technique pioneered by Pelham and Wang and refined by others, the role of substrate stiffness.

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