muramidase and ethyl-acetate

Psidium guajava L. is known to possess immune-modulatory properties in humans and other mammals. Although the positive effects of P. guajava-based diets on the immunological status have been shown for some fish species, the underlying molecular mechanisms of its protective effects remain to be investigated. The aims of this study were to evaluate the immune-modulatory effects of two guava fractions from dichloromethane (CC) and ethyl acetate (EA) on striped catfish with in vitro and in vivo experiments. Striped catfish head kidney leukocytes were stimulated with 40, 20, 10 and 0 μg/ml of each extract fraction, and the immune parameters (ROS, NOS, and lysozyme) were examined at 6 and 24 h post stimulation. A final concentration of each fraction at 40, 10 and 0 μg/fish was then intraperitoneally injected into the fish. After 6, 24, and 72 h of administration, immune parameters as well as the expression of some cytokines related to innate and adaptive immune responses, inflammation, and apoptosis were measured in the head kidney. Results indicated that the humoral (lysozyme) and cellular (ROS and NOS) immune endpoints were regulated differently by CC and EA fractions depending on dose and time in both, in vitro and in vivo experiments. With regards to the in vivo experiment, the CC fraction of the guava extract could significantly enhance the TLRs-MyD88-NF-κB signaling pathway by upregulating its cytokine genes (tlr1, tlr4, myd88, and traf6), following the upregulation of inflammatory (nfκb, tnf, il1β, and il6) and apoptosis (tp53 and casp8) genes 6 h after injection. Moreover, fish treated with both CC and EA fractions significantly enhanced cytokine gene expression including lys and inos at the later time points - 24 h or 72 h. Our observations suggest that P. guajava fractions modulate the immune, inflammatory, and apoptotic pathways.

Topics: Animals; Catfishes; Cytokines; Humans; Immunity; Mammals; Methylene Chloride; Muramidase; Myeloid Differentiation Factor 88; NF-kappa B; Plant Extracts; Psidium; Reactive Oxygen Species

PLGA particles have been extensively used as a sustained drug-delivery system, but there are multiple drawbacks when delivering proteins. The focus of this work is to address the most significant disadvantages to the W/O/W double emulsion procedure and demonstrate that simple changes to this procedure can have significant changes to particle size and dispersity and considerable improvements to protein loading, activity and sustained active protein release. A systematic approach was taken to analyze the effects of the following variables: solvent miscibility (dichloromethane (DCM), ethyl acetate, acetone), homogenization speed (10 000-25 000 rpm), PLGA concentration (10-30 mg/ml) and additives in both the organic (sucrose acetate isobutyrate (SAIB)) and aqueous (bovine serum albumin (BSA)) phases. Increasing solvent miscibility decreased particle size, dispersity and protein denaturation, while maintaining adequate protein loading. Increasing solvent miscibility also lowered the impact of homogenization on particle size and dispersity and protein activity. Changes to PLGA concentration demonstrated a minimum impact on particle size and dispersity, but showed an inverse relationship between protein encapsulation efficiency and particle protein weight percent. Most particles tested provided sustained release of active protein over 60 days. Increasing solvent miscibility resulted in increases in the percent of active protein released. When subjected to synthesis conditions with DCM as the solvent, BSA as a stabilizer resulted in the maximum stabilization of protein at a concentration of 100 mg/ml. At this concentration, BSA allowed for increases in the total amount of active protein delivered for all three solvents. The benefit of SAIB was primarily increased protein loading.

Topics: Acetates; Acetone; Animals; Cattle; Delayed-Action Preparations; Lactic Acid; Methylene Chloride; Muramidase; Nanoparticles; Nerve Regeneration; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Protein Denaturation; Serum Albumin, Bovine; Solubility; Solvents; Spinal Cord Injuries; Sucrose

The incorporation of the model protein hen egg white lysozyme into liquid in situ forming poly(lactide-co-glycolide) (PLGA) implant or microparticle formulations was investigated. Ternary solvent blends of dimethyl sulfoxide (DMSO), ethyl acetate and water were used to adjust the protein solubility in order to facilitate the incorporation of either dispersed or dissolved protein into the polymer solution. Lysozyme formed large gel particles when dispersed directly in the polymer solution. These formulations had a pronounced initial release. Non-aqueous precipitation of lysozyme from solutions in DMSO with ethyl acetate led to a reversible aggregation without loss in biological activity. Lysozyme could be incorporated in a finely dispersed state through an in situ precipitation by non-solvent or polymer addition. Non-aqueous precipitation could thus be utilized to manufacture biodegradable in situ forming drug delivery systems containing homogeneously distributed and bioactive protein.

Topics: Acetates; Chemistry, Pharmaceutical; Dimethyl Sulfoxide; Drug Delivery Systems; Lactic Acid; Micrococcus; Muramidase; Nanoparticles; Nephelometry and Turbidimetry; Particle Size; Photons; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Proteins; Solvents; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Water

The objective of this study was to investigate formulation and process parameters affecting protein encapsulation into PLGA microspheres during an ethyl acetate-based double emulsion microencapsulation process. Lysozyme was used as a model protein throughout this study. An aqueous lysozyme solution was emulsified in ethyl acetate containing 0.6 approximately 1.2 g PLGA75 : 25. The primary emulsion was then transferred quickly to an aqueous phase to make a water-in-oil-in-water emulsion. Ethyl acetate quenching was performed on the double emulsion stirred for 5, 15, 30 or 45 min. The resultant microspheres were further hardened, collected and dried overnight under vacuum. The bicinchoninic acid assay was carried out to determine the quantity of lysozyme present in the aqueous continuous phase and inside the microspheres. While the primary emulsion was stirred without quenching, lysozyme in the inner water phase continued diffusing across the ethyl acetate phase into the aqueous continuous phase. Emulsion droplets were also broken into smaller ones with ongoing stirring; this event also contributed to lysozyme leaking out of the inner water phase. The amount of lysozyme leaching to the aqueous continuous phase ranged from 4.79 +/- 2.1 to 51.9 +/- 5.3% under the experimental condition. Ethyl acetate quenching stopped the primary emulsion droplets from being fragmented into smaller ones and caused PLA75 : 25 precipitation to form microspheres. As a result, the rate of ethyl acetate removal influenced lysozyme encapsulation efficiency, as well as microsphere size. Depending on the timing of ethyl acetate quenching, lysozyme encapsulation efficiencies were found to be 9.89 +/- 4.53 approximately 75.82 +/- 6.55%. Optimization of the onset of ethyl acetate quenching and formulations could permit attainment of a desirable protein encapsulation efficiency.

Topics: Acetates; Biocompatible Materials; Drug Compounding; Emulsions; Lactic Acid; Microscopy, Electron, Scanning; Microspheres; Muramidase; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Proteins; Solubility; Solvents; Surface Properties; Time Factors; Water