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

Macromolecular approaches to prevent thrombosis and intimal hyperplasia following percutaneous coronary intervention.

Cardiovascular disease remains one of the largest contributors to death worldwide. Improvements in cardiovascular technology leading to the current generation of drug-eluting stents, bioresorbable stents, and drug-eluting balloons, coupled with advances in antirestenotic therapeutics developed by pharmaceutical community, have had a profound impact on quality of life and longevity. However, these procedures and devices contribute to both short- and long-term complications. Thus, room for improvement and development of new, alternative strategies exists. Two major approaches have been investigated to improve outcomes following percutaneous coronary intervention including perivascular delivery and luminal paving. For both approaches, polymers play a major role as controlled research vehicles, carriers for cells, and antithrombotic coatings. With improvements in catheter delivery devices and increases in our understanding of the biology of healthy and diseased vessels, the time is ripe for development of novel macromolecular coatings that can protect the vessel lumen following balloon angioplasty and promote healthy vascular healing

Development of affinity-based delivery of NGF from a chondroitin sulfate biomaterial.

Chondroitin sulfate is a major component of the extracellular matrix in both the central and peripheral nervous systems. Chondroitin sulfate is upregulated at injury, thus methods to promote neurite extension through chondroitin sulfate-rich matrices and synthetic scaffolds are needed. We describe the use of both chondroitin sulfate and a novel chondroitin sulfate-binding peptide to control the release of nerve growth factor. Interestingly, the novel chondroitin sulfate-binding peptide enhances the controlled release properties of the chondroitin sulfate gels. While introduction of chondroitin sulfate into a scaffold inhibits primary cortical outgrowth, the combination of chondroitin sulfate, chondroitin sulfate-binding peptide and nerve growth factor promotes primary cortical neurite outgrowth in chondroitin sulfate gels

An incubatable direct current stimulation system for in vitro studies of Mammalian cells.

The purpose of this study was to provide a simplified alternative technology and format for direct current stimulation of mammalian cells. An incubatable reusable stimulator was developed that effectively delivers a regulated current and does not require constant monitoring

Effects of a synthetic bioactive peptide on neurite growth and nerve growth factor release in chondroitin sulfate hydrogels.

Previous work has revealed robust dorsal root ganglia neurite growth in hydrogels of chondroitin sulfate. In the current work, it was determined whether addition of a synthetic bioactive peptide could augment neurite growth in these matrices via enhanced binding and sequestering of growth factors. Fluorescence recovery after photobleaching studies revealed that addition of peptide slowed nerve growth factor diffusivity in chondroitin sulfate gels, but not in control gels of hyaluronic acid. Furthermore, cultures of chick dorsal root ganglia in gels of hyaluronic acid or chondroitin sulfate revealed enhanced growth in chondroitin sulfate gels only upon addition of peptide. Taken together, these results suggest a synergistic nerve growth factor-binding activity between this peptide and chondroitin sulfate

Using Collagen Binding Poly(N-Isopropylacrylamide) Nanoparticles to Prevent Intravascular Platelet Adhesion and Activation

Balloon angioplasty, the most prevalent non-surgical treatment for Atherosclerosis, damages the endothelial layer of the artery, baring an underlying collagenous layer, which causes platelet adhesion and activation and eventual thrombosis and intimal hyperplasia. Previous work in our lab has used a collagen-binding peptidoglycan, dermatan-sulfate-SILY (DS-SILY), that has been shown to bind to type I collagen and prevent platelet adhesion and activation. Our goal is to fabricate nanoparticle-SILY by cross-linking SILY to a poly(N-isopropylacrylamide) (NIPAm) nanoparticle instead of a DS backbone, while retaining the SILY’s high collagen binding affinity and platelet inhibition capacity observed in DS-SILY. Using a biotin-streptavidin assay, we showed that nanoparticle-SILY has a high binding affinity when applied to a collagen-coated surface, starting at a concentration of 1 mg/mL and maximized at concentrations at or above 4 mg/mL. Using dynamic light scattering, we showed that the nanoparticle-SILY maintains the thermodynamic properties characteristic of NIPAm. The ability of the particles to inhibit platelet binding and activation will be tested by using an ELISA to measure NAP-2 and PF-4 expression in platelet rich plasma applied to a collagen surface treated with nanoparticle-SILY. We expect to find that nanoparticle-SILY is able to inhibit platelet activation, as evidenced by lower levels of NAP-2 and PF-4 released. If it can be shown that nanoparticle-SILY has the capability to prevent platelet adhesion and activation, future work could explore the potential of the nanoparticles to be loaded with anti-inflammatory peptides and used for the dual purpose of targeted drug delivery and collagen shielding

Proteoglycan Mimic of the Glycocalyx to Treat Endothelial Dysfunction

Patients with kidney failure usually undergo hemodialysis, a process by which toxins produced by the body are filtered from the blood, in order to survive. The preferred form for vascular access is called an arteriovenousfistula (AVF), a surgically created connection between an artery and vein that is utilized to undergo dialysis. However, AVFs have a failure rate of 50-60%. One of the contributions to AVF failure is endothelial cell dysfunction and loss of glycocalyx, which allows neutrophils and other native cells into the media of the vessel, which causes an inflammatory response. Our lab addresses endothelial dysfunction by mimicking the function of the glycocalyx to prevent transmigration of inflammatory cells and ultimately create a healthier vessel for hemodialysis. We have synthesized several glycocalyx mimics consisting of a dermatan sulfate backbone with multiple selectin and ICAM-binding peptides attached. Initial testing involved determining the ability of the variants to bind to inflamed endothelial cells. We also cultured human promyelocytic leukemia cells (HL60) and used retinoic acid to differentiate them into neutrophils. These cells would then test the glycocalyx mimics ability to prevent migration of neutrophils. Thus far, we have seen that the glycocalyx mimics binding to endothelial cells and that this binding is dependent upon the type of selectin and/or ICAM-binding peptides as well as how many peptides are present per dermatan sulfate backbone. We have also shown that proliferation occurs 10 days after seeding, and that rentinoic acid (RA) differentiates HL60 cells into neutrophils. We have developed a protocol for differentiation of HL60 cells to neutrophils, a promising set of glycocalyx mimics, and culturing method for HL60 cells

Suppression of osteoarthritis via molecular engineering of an aggrecan mimetic

Osteoarthritis (OA) progresses via a feed-forward cycle in which inflammation leads to the up-regulation of catabolic enzymes that cleave the extracellular matrix (ECM) components. Fragments of ECM molecules hyaluronic acid and collagen type II further stimulate inflammation. The degradation of aggrecan is a critical early event in OA due to aggrecan’s ability to protect other ECM components from degradation and support the compressive strength of cartilage. Characterized herein is an aggrecan mimic’s (mAGC) ability to replace the functions of native aggrecan and halt the progression of OA. We examine mAGC in both ex vivo cartilage tissue models and in vivo animal models. Aggrecan-depleted cartilage plugs had only ~30% of the compressive strength of intact plug. mAGC was able to diffuse into the cartilage tissue and restore the compressive strength to 90% of the intact healthy cartilage. Depletion of aggrecan also resulted in an increase in catabolic gene expression by chondrocytes that was further amplified with additional inflammatory stimuli. Treatment with mAGC resulted in chondrocyte gene expression of catabolic enzymes at the same lower levels as healthy intact cartilage, both with and without inflammatory stimulation over 21 days. Intact cartilage plugs exposed to osteoarthritic synovial fluid resulted in high degradation of ECM components as measured by release into the culture media over an 8-day period. A single pretreatment with mAGC decreased this degradation to levels similar to those in healthy cultured controls. Further, inflammation and catabolic enzymatic gene expression was lowered in treated plugs to near healthy levels, even in the presence of the inflammatory and enzyme-rich synovial fluid. The data indicates that by providing robust protection against degradation and restoring the mechanical environment, the pro-inflammatory signals that cause upregulation of the degrading enzymes are decreased. In an aggressive rat model, mAGC was able to keep catabolic enzyme levels closer to healthy levels, preserve the proteoglycan content of cartilage tissue, and decrease bone loss when compared with untreated controls. In a nontraumatic guinea pig model, mACG suppressed the progression of OA. This study provides the ground-work for development of an intra-articular therapy that reduces fragmentation of key extracellular matrix components and restores the mechanical environment of the cartilage tissue, resulting in decrease in inflammation and catabolic enzyme production. The therapy has the potential to promote a healthy environment for future tissue regeneration