Hyaluronan for Regenerative Medicine Applications: A Macrophage-Centered Approach

Biomaterials with the innate ability to inhibit inflammation and promote tissue integration and angiogenesis show positive outcomes in regenerative medicine. Of the cells that contribute toward the outcome of the biomaterials, macrophages are one of the most influential. Macrophages are highly plastic host immune cells, which persist for the lifetime of the biomaterial and have the robust ability to influence inflammation, tissue integration, and angiogenesis dependent on their polarization. Developing biomaterials that elicit desired macrophage polarization is a key component to their success in the host environment. Hyaluronan (HA) has been extensively explored in regenerative medicine due to its natural derivation and favorable mechanical properties. Biologically, HA exists in a variety of molecular weights in the soluble form and may become crosslinked during inflammatory events. We explored the influence of soluble high and low molecular weight HAs as well as crosslinked HA on macrophage polarization in cytokine naïve and activated (classical or alternative) macrophages. Soluble low molecular weight HA (LMWHA) was found to stimulate classical activation of the macrophage, promoting inflammation, tissue destructive, and anti-angiogenic functions of the macrophage. On the other hand, high molecular weight HA (HMWHA) promoted tissue integrative, anti-inflammatory, and pro-angiogenic functions of macrophages, suggesting it polarized and reprogramed macrophages to the alternatively activated form. We exploited the favorable interactions of HMWHA with macrophages to bioengineer hydrogel scaffolds suitable for regenerative medicine purposes. These hydrogels exhibited macrophage-polarizing properties dependent on the molecular weight between crosslinks. HA hydrogels with high molecular weight between crosslinks retained the alternatively activating effects of soluble HMWHA, whereas those with low molecular weight between crosslinks elicited characteristics of both classically and alternatively activated macrophages. Our study suggested that HMWHA retains properties favorable for regenerative medicine when it is loosely crosslinked. Prior to this study, macrophage polarization and reprogramming from soluble and crosslinked HA had never determined. Beyond regenerative medicine, the understanding of HA-macrophage interactions has implications in rheumatology, sterile inflammation, and cancer. Our study supports further understanding of biomaterial macrophage interactions to derive therapeutic benefit

In-house preparation of hydrogels for batch affinity purification of glutathione <it>S</it>-transferase tagged recombinant proteins

<p>Abstract</p> <p>Background</p> <p>Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins.</p> <p>Results</p> <p>Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of <it>E. coli</it> lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads.</p> <p>Conclusions</p> <p>GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.</p

Reducible Micelleplexes are Stable Systems for Anti-miRNA Delivery in Cerebrospinal Fluid

Glioblastoma multiforme (GBM) and other central nervous system (CNS) cancers have poor long-term prognosis, and there is a significant need for improved treatments. GBM initiation and progression are mediated, in part, by microRNA (miRNA), which are endogenous posttranscriptional gene regulators. Misregulation of miRNAs is a potential target for therapeutic intervention in GBM. In this work, a micelle-like nanoparticle delivery system based upon the block copolymer poly­(ethylene glycol-<i>b</i>-lactide-<i>b</i>-arginine) was designed with and without a reducible linkage between the lactide and RNA-binding peptide, R₁₅, to assess the ability of the micelle-like particles to disassemble. Using confocal live cell imaging, intracellular dissociation was pronounced for the reducible micelleplexes. This dissociation was also supported by higher efficiency in a dual luciferase assay specific for the miRNA of interest, miR-21. Notably, micelleplexes were found to have significantly better stability and higher anti-miRNA activity in cerebrospinal fluid than in human plasma, suggesting an advantage for applying micelleplexes to CNS diseases and in vivo CNS therapeutics. The reducible delivery system was determined to be a promising delivery platform for the treatment of CNS diseases with miRNA therapy

High and Low Molecular Weight Hyaluronic Acid Differentially Influence Macrophage Activation

Macrophages exhibit phenotypic diversity permitting wide-ranging roles in maintaining physiologic homeostasis. Hyaluronic acid, a major glycosaminoglycan of the extracellular matrix, has been shown to have differential signaling based on its molecular weight. With this in mind, the main objective of this study was to elucidate the role of hyaluronic acid molecular weight on macrophage activation and reprogramming. Changes in macrophage activation were assessed by activation state selective marker measurement, specifically quantitative real time polymerase chain reaction, and cytokine enzyme-linked immunoassays, after macrophage treatment with differing molecular weights of hyaluronic acid under four conditions: the resting state, concurrent with classical activation, and following inflammation involving either classically or alternatively activated macrophages. Regardless of initial polarization state, low molecular weight hyaluronic acid induced a classically activated-like state, confirmed by up-regulation of pro-inflammatory genes, including <i>nos2</i>, <i>tnf</i>, <i>il12b</i>, and <i>cd80,</i> and enhanced secretion of nitric oxide and TNF-α. High molecular weight hyaluronic acid promoted an alternatively activated-like state, confirmed by up regulation of pro-resolving gene transcription, including <i>arg1</i>, <i>il10</i>, and <i>mrc1,</i> and enhanced arginase activity. Overall, our observations suggest that macrophages undergo phenotypic changes dependent on molecular weight of hyaluronan that correspond to either (1) pro-inflammatory response for low molecular weight HA or (2) pro-resolving response for high molecular weight HA. These observations bring significant further understanding of the influence of extracellular matrix polymers, hyaluronic acid in particular, on regulating the inflammatory response of macrophages. This knowledge can be used to guide the design of HA-containing biomaterials to better utilize the natural response to HAs

Proteolytically activated anti-bacterial hydrogel microspheres

Hydrogels are finding increased clinical utility as advances continue to exploit their favorable material properties. Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectious surfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favorable material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure the materials are non-infectious upon placement, but sterilization is not absolute and infections are still expected. Natural, anti-bacterial proteins have been discovered which have the potential to act as anti-infectious agents; however, the proteins are toxic and need localized release to have therapeutic efficacy without toxicity. In these studies, we explore the use of the glutathione s-transferase (GST) to anchor the bactericidal peptide, melittin, to the surface of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We show that therapeutic levels of protein can be anchored to the surface of the microspheres using the GST anchor. We compared the therapeutic efficacy of recombinant melittin released from PEGDA microspheres to melittin. We found that, when released by an activating enzyme, thrombin, recombinant melittin efficiently inhibits growth of the pathogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. We conclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres and the protein will remain immobilized under physiological conditions until the protein is enzymatically released