muramidase and methacrylic-acid

Bioprinting of living cells is rapidly developing as an advanced biofabrication approach to engineer tissues. Bioinks can be extruded in three-dimensions (3D) to fabricate complex and hierarchical constructs for implantation. However, a lack of functionality can often be attributed to poor bioink properties. Indeed, advanced bioinks encapsulating living cells should: (i) present optimal rheological properties and retain 3D structure post fabrication, (ii) promote cell viability and support cell differentiation, and (iii) localise proteins of interest (e.g. vascular endothelial growth factor (VEGF)) to stimulate encapsulated cell activity and tissue ingrowth upon implantation. In this study, we present the results of the inclusion of a synthetic nanoclay, Laponite

Topics: Animals; Bioprinting; Cattle; Cell Proliferation; Cell Survival; Chickens; Chorioallantoic Membrane; Gelatin; Humans; Hydrogels; Ink; Mesenchymal Stem Cells; Methacrylates; Muramidase; Nanocomposites; Neovascularization, Physiologic; Osteogenesis; Porosity; Serum Albumin, Bovine; Silicates; Swine; Vascular Endothelial Growth Factor A

Designing structure and morphology of macroporous hydrogels is crucial for their applications in controlled release systems of macromolecular drugs. Macroporous hydrogels, consisting of methacrylic acid (MAA) and either acryl amide (AAm) or 2-hydroxyethyl methacrylate (HEMA) (1st network), were prepared for this purpose by cryogelation (single network cryogels, SNCs). Macroporous interpenetrating polymer network (IPN) hydrogel composites were then prepared by a sequential strategy, the 2nd network consisting of chitosan (CS) cross-linked with poly(ethyleneglycol) diglycidyl ether (PEGDGE) being generated by the sorption of a CS and PEGDGE mixture in the 1st network followed by cross-linking. A strong difference in the behavior of SNCs and IPN hydrogel composites was found during the loading and release of lysozyme (LYS) used as macromolecular drug model. Thus, while the amount of LYS loaded on SNCs was higher than that loaded on the IPNs, the release of LYS from SNCs occurred at pH 2, when the ratio between MAA and AAm was 50:50, and only at pH 1 when the ratio between MAA and AAm was 70:30. The 2nd network led to the decrease of the pore size of the IPNs, mainly when the initial concentration of monomers was 10wt/v%, but the presence of CS facilitates the LYS release from IPNs, mainly at a concentration of monomer of 5wt/v%, and when HEMA was used as nonionic comonomer.

Topics: Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Drug Compounding; Drug Liberation; Hydrogels; Hydrogen-Ion Concentration; Kinetics; Methacrylates; Muramidase; Polyethylene Glycols; Porosity

The aim of this study was to determine the influence of hyaluronic acid (HA) on lysozyme sorption in model contact lenses containing varying amounts of methacrylic acid (MAA). One model conventional hydrogel (poly(2-hydroxyethyl methacrylate) (pHEMA)) and two model silicone hydrogels (pHEMA, methacryloxypropyltris(trimethylsiloxy)silane (pHEMA TRIS) and N,N-dimethylacrylamide, TRIS (DMAA TRIS)) lens materials were prepared with and without MAA at two different concentrations (1.7 and 5%). HA, along with dendrimers, was loaded into these model contact lens materials and then cross-linked with 1-ethyl-3-(3-dimethylamino propyl)-carbodiimide (EDC). Equilibrium water content (EWC), advancing water contact angle and lysozyme sorption on these lens materials were investigated. In the HA-containing materials, the presence (P < 0.05) and amount (P < 0.05) of MAA increased the EWC of the materials. For most materials, addition of MAA reduced the advancing contact angles (P < 0.05) and for all the materials, the addition of HA further improved hydrophilicity (P < 0.05). For the non-HA containing hydrogels, the presence (P < 0.05) and amount (P < 0.05) of MAA increased lysozyme sorption. The presence of HA decreased lysozyme sorption for all materials (P < 0.05). MAA appears to work synergistically with HA to increase the EWC in addition to improving the hydrophilicity of model pHEMA-based and silicone hydrogel contact lens materials. Hydrogel materials that contain HA have tremendous potential as hydrophilic, protein-resistant contact lens materials.

Topics: Adsorption; Contact Lenses; Hyaluronic Acid; Hydrophobic and Hydrophilic Interactions; Methacrylates; Muramidase; Surface Properties; Water

Molecular imprinting is a method to fabricate a polymeric material (molecularly imprinted polymer or MIP) capable of selectively recognizing template molecules. Molecular imprinting of small molecules has been studied widely. Less common, however, is the imprinting of biological macromolecules, including proteins, among which lysozyme is an important molecule in the food, pharmaceutical, and diagnostic sciences. In this study, lysozyme MIP was fabricated in two steps. First, lysozyme, PEG600DMA, and methacrylic acid were used as the template molecule, cross-linking monomer, and the functional monomer, respectively, in a UV free-radical polymerization process to synthesize a polymeric gel. Second, lysozyme was removed by enzymatic digestion. Non-imprinted polymer (NIP) was synthesized without lysozyme addition. To evaluate the preferential binding capability of MIP, lysozyme, RNase A, or a 50:50 mixture of lysozyme and RNase A was added to MIP and NIP and then released by digestion. It was found that when more lysozyme was added to the reaction mixture, the quantity of protein released from the polymer increased, reflecting more potential binding sites. Tests of MIP with a competitive binding mixture of lysozyme and RNase A showed the MIP preferentially bound a greater amount of lysozyme, up to 20 times more than RNase A. NIP bound only small amounts of both proteins and did not show a preference for binding either lysozyme or RNase A. These results demonstrate that lysozyme was successfully imprinted into the MIP by UV free-radical polymerization, and the fabricated MIP was able to preferentially bind its template protein.

Topics: Animals; Chickens; Cross-Linking Reagents; Methacrylates; Molecular Imprinting; Muramidase; Polyethylene Glycols; Polymerization; Protein Binding

The wettability of poly[2-hydroxyethyl methacrylate-co-methacrylic acid] (pHEMA-MAA) soft contact lenses was investigated in the absence and presence of block copolymer surfactants and lysozyme using the sessile drop method. The advancing dynamic contact angles (Thetaw/a) values are reported for water as a function of sequential wetting and drying cycles. The Thetaw/a values for the pHEMA-MAA in the absence of surfactant and lysozyme increased from approximately 20 degrees to 100 degrees as the number of cycles increased from two to ten, and they were independent of the pHEMA-MAA bulk water content. The change from the highly hydrophilic to hydrophobic pHEMA-MAA surface could not be reversed using the sequential wetting and drying cycles even under repeated exposures to saline solution. The effect of block copolymer surfactants with different molecular weights (MW) and hydrophilic-lipophilic balance (HLB) values on the pHEMA-MAA wettability were also studied. Low Theta(w/a) values were observed for pHEMA-MAA hydrogels that were treated with T1304 (MW 10500, HLB 14) and T904 (MW 6700, HLB 15). The surface tension data indicated that these surfactants were incompletely desorbed from the pHEMA-MAA and that the rate of desorption was slow in the timescale of the cycling experiments. Comparatively, poor wettability was observed for pHEMA-MAA surfaces presoaked in T304 (MW 1650, HLB 16) and T1107 (MW 15000, HLB 24) as Thetaw/a values greater than 90 degrees were measured for these surfactants. The surface tension data indicated that the rate of desorption of T304 and T1107 from the pHEMA-MAA was rapid and that they had a low affinity to the pHEMA-MAA. High contact angles were observed for the pHEMA-MAA hydrogels treated with lysozyme and also for the T1107 presoaked pHEMA-MAA that was also treated with lysozyme. Zero wetting angles throughout the sequential cycling were observed for the T1304 pre-treated pHEMA-MAA that had been treated with lysozyme. These results suggested that the adsorbed lysozyme on the pHEMA-MAA hydrogel had no significant influence on its wetting properties when the hydrogel was pre-treated with T1304.

Topics: Contact Lenses, Hydrophilic; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Methacrylates; Muramidase; Polyhydroxyethyl Methacrylate; Polymers; Spectrophotometry; Surface Tension; Surface-Active Agents; Temperature; Time Factors; Water; Wettability

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.

Topics: Acetylglucosamine; Acrylamides; Acrylates; Animals; Binding Sites; Chemical Precipitation; Egg White; Hydrogen-Ion Concentration; Ions; Methacrylates; Muramidase; Osmolar Concentration; Polymers; Solutions; Succinates

To examine the effect of hydrogel lens monomer constituents on protein sorption.. A series of hydroxyethylmethacrylate (HEMA)-based hydrogels with various amounts of methacrylic acid (MAA) or N-vinyl pyrrolidone (NVP) were synthesized. A radiolabel tracer technique was used to measure the amount of protein adsorbed on or penetrating into the hydrogels. Penetration of fluorescence-labeled proteins in the hydrogels was studied by laser scanning confocal microscopy. Single-protein solutions of human serum albumin (HSA) and hen egg lysozyme were studied.. Inclusion of the comonomers MAA or NVP in hydrogels resulted in an increase in water content and also had a strong impact on protein sorption. An increase in the amount of MAA in the poly(HEMA-co-MAA) hydrogels increased lysozyme adsorption and penetration but reduced HSA adsorption. However, the amount of protein adsorbed for both HSA and lysozyme increased with the amount of NVP in the poly(HEMA-co-NVP) hydrogels. In contrast to the marked effect of MAA on protein sorption, in particular, on lysozyme sorption, NVP had little influence on protein sorption. When a hydrogel contains both MAA and NVP, MAA has the dominant effect on protein sorption-in particular, on lysozyme sorption. Furthermore, a large difference was observed in the amount of lysozyme adsorbed on the hydrogels that had similar water contents but little variation in adsorption of HSA.. Negatively charged carboxyl groups of the MAA constituent may influence lysozyme sorption in two ways: by electrostatic attraction and by increasing the possibility for the small lysozyme molecule to penetrate the hydrogels. Interactions of the surface lactam groups of NVP with proteins may be attributable to the attraction of proteins to NVP. Water content is not a primary factor in determining protein adsorption. It appears that the monomer constituents, such as MAA or NVP, control protein adsorption.

Topics: Adsorption; Contact Lenses; Methacrylates; Microscopy, Confocal; Muramidase; Polyhydroxyethyl Methacrylate; Protein Binding; Pyrrolidinones; Serum Albumin; Static Electricity