Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.

In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy.

Characterization of endocytic uptake of MK2-inhibitor peptides.

Cell penetrating peptides (CPP) have been widely used to increase the cellular delivery of their associated cargo. Multiple modes of uptake have been identified; however, they cannot be predicted a priori. Elucidating these mechanisms is important for understanding peptide function as well as further optimizing cellular delivery. We have developed a class of mitogen activated protein kinase activated protein kinase 2 (MK2) inhibitor peptides, named FAK and YARA that utilize CPP domains to gain cellular access. In this study, we investigate the mechanism of endocytosis of these MK2 inhibitors by examining the uptake of fluorescently labeled peptide in human monocyte (THP-1) and mesothelial cells, and looking for colocalization with known markers of endocytosis. Our results indicate that uptake of the MK2 inhibitors was minimally enhanced by the addition of the fluorescent label, and that the type of endocytosis used by the inhibitor depends on several factors including concentration, cell type, and which CPP was used. We found that in THP-1 cells, the uptake of YARA occurred primarily via macropinocytosis, whereas FAK entered via all three mechanisms of endocytosis examined in this study. In mesothelial cells, uptake of YARA occurred via caveolae-mediated endocytosis, but became less specific at higher concentrations; whereas uptake of FAK occurred through clathrin-mediated endocytosis. In all cases, the delivery resulted in active inhibition of MK2. In summary, the results support endocytic uptake of fluorescently labeled FAK and YARA in two different cell lines, with the mechanism of uptake dependent on extracellular concentration, cell type, and choice of CPP.

Synthesis and characterization of a poly(lactic-co-glycolic acid) core + poly(N-isopropylacrylamide) shell nanoparticle system.

Poly(lactic-co-glycolic acid) (PLGA) is a popular material used to prepare nanoparticles for drug delivery. However, PLGA nanoparticles lack desirable attributes including active targeting abilities, resistance to aggregation during lyophilization, and the ability to respond to dynamic environmental stimuli. To overcome these issues, we fabricated a nanoparticle consisting of a PLGA core encapsulated within a shell of poly(N-isopropylacrylamide). Dynamic light scattering and transmission electron microscope imaging were used to characterize the nanoparticles, while an MTT assay and ELISA suggested biocompatibility in THP1 cells. Finally, a collagen type II binding assay showed successful modification of these nanoparticles with an active targeting moiety.

Cell-penetrating peptides can confer biological function: regulation of inflammatory cytokines in human monocytes by MK2 inhibitor peptides.

Cell-penetrating peptides have been used as a method of delivering biologically active peptide for over two decades. In this paper, we covalently attached four different cell-penetrating peptides to a peptide that inhibits a kinase important in inflammation, mitogen-activated protein kinase activated protein kinase 2 (MAPKAP2 or MK2). We evaluated the specificity, toxicity, and functionality of these therapeutics in an in vitro model of inflammation using THP-1 monocytes. When treated with the MK2 peptide inhibitors, activated THP-1 human monocytes challenged with lipopolysaccharide (LPS) showed a decrease in TNF-α and IL-6 excretion without apparent toxicity. In addition, western blot analysis revealed decreases in the phosphorylation of heat shock protein 27 (HSP27), a downstream substrate of MK2. These results suggested that our peptides inhibited MK2 activity in vitro and should be investigated further as a potential therapeutic for applications involving inflammation. Furthermore, our results suggested that cell-penetrating peptides can be bioactive.

Peptide inhibitors of MK2 show promise for inhibition of abdominal adhesions.

BACKGROUND: Abdominal adhesions are a common side effect of surgical procedures with complications including infertility, chronic pain, and bowel obstruction, which may lead to the need for surgical lyses of the adhesions. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) has been implicated in several diseases, involving inflammation and fibrosis. Thus, the development of a cell-penetrating peptide (CPP) that modulates MK2 activity may confer therapeutic benefit after abdominal surgery in general and more specifically after bowel anastomosis. METHODS: This study evaluated the function of a CPP inhibitor of MK2 in human mesothelial cells and in a rat bowel anastomosis model. To determine IC50 and basic specificity, kinase inhibition was performed using a radiometric assay. Enzyme-linked immunoassay (ELISA) was used to evaluate interleukin-6 (IL-6) expression in response to IL-1ß and tumor necrosis factor-α (TNF-α) stimulation in vitro to validate MK2 kinase inhibition. Following bowel anastomosis (10 rats for each control and treatment at 4 and 10 d), the rats were evaluated for weight loss, normal healing (colonic burst strength and hydroxyproline content at the anastomosis), and number and density of adhesions. RESULTS: The IC50 of the MK2 inhibitor peptide (22 µM) was similar to that of the nonspecific small molecule rottlerin (IC50 = 5 µM). The MK2 inhibitor peptide was effective at suppressing IL-1ß and TNF-α stimulated IL-6 expression in mesothelial cells. In vivo, the MK2 inhibitor peptide was effective at suppressing both the density and number of adhesions formed as a result of bowel an anastamosis. Importantly, the peptide had no negative effect on normal healing. CONCLUSIONS: In conclusion, the peptide inhibitor of MK2, MMI-0100, has the potential to significantly reduce inflammation through suppression of inflammatory cytokine expression and showed promise as a therapeutic for abdominal adhesions.

Cell penetrating peptides can exert biological activity: a review.

Cell penetrating peptides (CPPs) have been successful in delivering cargo into many different cell types and are an important alternative to other methods of permeation that might damage the integrity of the cell membrane. The traditional view of CPPs is that they are inert molecules that can be successfully used to deliver many cargos intracellularly. The goal of this review is to challenge this traditional understanding of CPPs. Recent literature has demonstrated that CPPs themselves can convey biological activity, including the alteration of gene expression and inhibition of protein kinases and proteolytic activity. Further characterization of CPPs is required to determine the extent of this activity. Research into the use of CPPs for intracellular delivery should continue with investigators being aware of these recent results.

The development of a tissue-engineered cornea: biomaterials and culture methods.

The field of corneal tissue engineering has made many strides in recent years. The challenges of engineering a biocompatible, mechanically stable, and optically transparent tissue are significant. To overcome these challenges, researchers have adopted two basic approaches: cell-based strategies for manipulating cells to create their own extracellular matrix, and scaffold-based strategies for providing strong and transparent matrices upon which to grow cells. Both strategies have met with some degree of success. In addition, recent advances have been made in innervating a tissue-engineered construct. Future work will need to focus on further improving mechanical stability of engineered constructs as well as improving the host response to implantation.