Properties of superporous hydrogels for drug delivery

The purpose of the present study was to investigate the properties of a novel hydrogel system, the superporous hydrogel. Superporous hydrogels have been developed over the last several years as a drug delivery system. Studies on superporous hydrogels made of poly(acrylic acid-co-acrylamide) were focused on the properties that will be useful in oral delivery for gastric retention and vascular implantation as a chemoembolization device. Superporous hydrogels have a porous structure that acts as a capillary network. The capillary properties of superporous hydrogels are a result of an oriented pore structure that is produced during the production. High interconnectivity is present in the majority of the superporous hydrogels, but at the surface, little interconnectivity is present. The high interconnectivity and pore structure is maintained when the superporous hydrogel is compressed radially, but the interconnection is disrupted when compression is axial. A reduction in dry volume of 80% is possible, allowing the superporous hydrogel to be small enough to be taken as an oral dosage form. The swelling of the compressed superporous hydrogels is slightly slower than the uncompressed superporous hydrogels but still much faster than a similar nonporous hydrogel. The swelling of superporous hydrogels is responsive to the environment in which the swelling takes place. In acidic conditions, superporous hydrogels swell to a much lower degree than in basic solutions because of the ionization of the acrylic acid on the polymer chains. The swollen size of the superporous hydrogel is large enough to be maintained in the stomach after ingestion. The density of the superporous hydrogel causes it to float in simulated gastric fluid, which will aid in the retention in the stomach prior to swelling. The bioadhesive properties of poly(acrylic acid) are also expected to increase retention of the superporous hydrogel at early times. Swelling of superporous hydrogels in blood is very slow due to the poor wetting of the dry hydrogel by blood and the viscous properties of blood. Plasma and serum both wet the dry hydrogel and swelled the superporous hydrogel at a much faster rate. The properties of the superporous hydrogels indicate that it will be very applicable to oral administration and vascular implantation

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

Decellularized Human Cornea for Reconstructing the Corneal Epithelium and Anterior Stroma

In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro, while corneas treated with SDS supported the growth of only fibroblasts and not epithelial cells. Decellularized human corneas provide a scaffold that can support the growth of corneal epithelial cells and stromal fibroblasts. This approach may be useful for reconstructing the anterior cornea and limbus using autologous cells

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

SEM images show dehydrated PHEMA-PEGDA.

<p>Water (A), sucrose (B), and benzyl alcohol (C) were used as porogens. Low magnification SEM images are displayed in the top row. Corresponding SEM images with higher magnification are shown in the bottom row. Image analysis was used to measure pore areas. Histograms display pore areas for the scaffolds made with water (D, n = 558), sucrose (E, n = 12,533), or benzyl alcohol (F, n = 11,485) porogens.</p

NMR spectroscopy results indicate that PEG chains appear to have leached out of the PHEMA-PEGDA scaffold following 3 days soaking in PBS.

<p>The signal for the middle O-CH2 group of PEGDA (A) and the middle O-CH2 group of PEG (B) were both found at approximately 70 ppm. Signals that represent the repeating side groups in PHEMA were not observed in PBS, however, indicating neither HEMA nor PHEMA leached out.</p

Tensile testing showed that the porogens can significantly alter the mechanical properties.

<p>The PHEMA-PEGDA made with water porogen was significantly stiffer than samples made with benzyl alcohol porogen. Low damping factors (tan (δ)) show that the PHEMA-PEGDA samples are viscoelastic with dominant elastic properties (B; n = 3). Tension was applied until rupture to determine the ductility and overall strength of the scaffolds (C). “X” marks the failure or rupture point of each sample. Horizontal bars identify samples with a statistically significant difference (p<0.05).</p

Toroidal-Spiral Particles for Codelivery of Anti-VEGFR‑2 Antibody and Irinotecan: A Potential Implant to Hinder Recurrence of Glioblastoma Multiforme

Heterogeneous toroidal-spiral particles (TSPs) were generated by polymer droplet sedimentation, interaction, and cross-linking. TSPs provide a platform for encapsulation and release of multiple compounds of different sizes and physicochemical properties. As a model system, we demonstrate the encapsulation and independently controlled release of an anti-VEGFR-2 antibody and irinotecan for the treatment of glioblastoma multiforme. The anti-VEGFR-2 antibody was released from the TS channels and its binding to HUVECs was confirmed by confocal microscopy and flow cytometry, suggesting active antibody encapsulation and release. Irinotecan, a small molecule drug, was released from the dense polymer matrix of poly­(ethylene glycol) diacrylate (MW ∼ 700 g/mol; PEGDA 700). Released irinotecan inhibited the proliferation of U251 malignant glioma cells. Since the therapeutic compounds are released through different pathways, specifically diffusion through the polymer matrix versus TS channels, the release rate can be controlled independently through the design of the structure and material of particle components