Myoglobin diet affected the colonic mucus layer and barrier by increasing the abundance of several beneficial gut bacteria.

The study aimed to explore the in vitro digestion of a myoglobin diet and its relationship with the gut microbiota and intestinal barrier at two feeding time points. The in vitro study indicated that myoglobin diets had a higher α-helix content and lower digestibility. The particle sizes of the digested myoglobin diets were higher but their zeta potential values were lower than those of a casein diet. C57BL/6J mice were fed with a casein diet incorporated with 0, 0.38%, 1.13% and 3.39% myoglobin for 3 and 8 weeks. With the increase of the myoglobin content, the relative abundance of Akkermansia muciniphila increased significantly. Moreover, the content of short chain fatty acids tended to increase and then decrease as the myoglobin content increased at 3 weeks. Furthermore, the low-myoglobin diet upregulated the gene expression involved in colonic mucin and tight junction proteins by increasing the proportion of beneficial microbiota. However, the high-myoglobin diet had adverse effects.

Confinement Facilitated Protein Stabilization As Investigated by Small-Angle Neutron Scattering.

While mesoporous silicas have been shown to be a compelling candidate for drug delivery and the implementation of biotechnological applications requiring protein confinement and immobilization, the understanding of protein behavior upon physical adsorption into silica pores is limited. Many indirect methods are available to assess general adsorbed protein stability, such as Fourier-transform infrared spectroscopy and activity assays. However, the limitation of these methods is that spatial protein arrangement within the pores cannot be assessed. Mesoporous silicas pose a distinct challenge to direct methods, such as transmission electron microscopy, which lacks the contrast and resolution required to adequately observe immobilized protein structure, and nuclear magnetic resonance, which is computationally intensive and requires knowledge of the primary structure a priori. Small-angle neutron scattering can surmount these limitations and observe spatial protein arrangement within pores. Hereby, we observe the stabilization of fluid-like protein arrangement, facilitated by geometry-dependent crowding effects in cylindrical pores of ordered mesoporous silica, SBA-15. Stabilization is induced from a fluid-like structure factor, which is observed for samples at maximum protein loading in SBA-15 with pore diameters of 6.4 and 8.1 nm. Application of this effect for prevention of irreversible aggregation in high concentration environments is proposed.

Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.

The phenomenon known as renal "ischemic preconditioning," whereby an initial ischemic insult induces resistance against subsequent kidney damage, has been well established in the experimental literature. However, a clinically applicable way to safely recapitulate this state has not been defined. We hypothesized that a unique combination of agents (nitrited myoglobin [N-Mgb] + tin protoporphyrin [SnPP]) can achieve these ends safely and synergistically, increasing cytoprotective proteins (eg, heme oxygenase 1 [HO-1], interleukin 10 [IL-10], and haptoglobin) in kidney cells. To test this hypothesis, CD-1 mice received 1 mg of N-Mgb and 1 µmol of SnPP, either alone or in combination. Renal cortical HO-1, haptoglobin, and IL-10 gene expressions (messenger RNA [mRNA], protein levels) were determined 4 and 18 hours later. Cytoresistance to 3 forms of acute kidney injury (AKI; glycerol-induced rhabdomyolysis, maleate nephrotoxicity, and postischemic AKI progression to chronic kidney disease [CKD]) was assessed. To ascertain whether cytoresistance might emerge in extrarenal organs, hepatic HO-1, IL-10, and haptoglobin levels were also measured, and resistance to 25 minutes of hepatic ischemia-reperfusion injury and hepatotoxicity (intraperitoneal glycerol injection) was sought. N-Mgb + SnPP induced additive or synergistic increases in renal HO-1, haptoglobin, and IL-10 mRNA and protein levels (up to 20-fold) without inducing any apparent renal or extrarenal damage. After 18 hours of post-treatment, marked or complete protection against glycerol-induced AKI, maleate-induced AKI, and postischemic AKI progression to CKD had emerged. Combined N-Mgb + SnPP was more protective than either agent alone (assessed in glycerol model). N-Mgb + SnPP also upregulated cytoprotective pathways in liver and induced marked protection against both hepatic ischemia-reperfusion and toxic liver damage. In conclusion, we posit that "preconditioning" with combined administration of N-Mgb + SnPP represents a promising approach for protecting against diverse forms of renal and nonrenal (hepatic) forms of tissue damage.

New biodegradable dextran-based hydrogels for protein delivery: Synthesis and characterization.

A new derivative of dextran grafted with polyethylene glycol methacrylate through a carbonate bond (DEX-PEG-MA) has been synthesized and characterized. The photo-crosslinking reaction of DEX-PEG-MA allowed the obtainment of biodegradable networks tested for their mechanical and release properties. The new hydrogels were compared with those made of dextran methacrylate (DEX-MA), often employed as drug delivery systems of small molecules. The inclusion of PEG as a spacer created additional interactions among the polymeric chains improving the extreme fragility and lack of hardness typical of gels made of DEX-MA. Moreover, the different behavior in terms of swelling and degradability of the networks was able to affect the release of a model macromolecule over time, making DEX-PEG-MA matrices suitable candidates for the delivery of high molecular weight peptides. Interestingly, the combination of the two dextran derivatives showed intermediate ability to modulate the release of high molecular weight macromolecules.

Tunable protein release from acetalated dextran microparticles: a platform for delivery of protein therapeutics to the heart post-MI.

The leading cause of death in the United States is cardiovascular disease. The majority of these cases result from heart failure post-myocardial infarction (MI). We present data providing evidence for use of acetalated dextran (AcDex) microparticles as a delivery vehicle for therapeutics to the heart post-MI. We harnessed the tunable degradation and acid-sensitivity of AcDex in the design of microparticles for intramyocardial injection. The particles released a model protein, myoglobin, and a sensitive growth factor, basic fibroblast growth factor (bFGF), over a wide range of time frames (from days to weeks) based on the percentage of cyclic acetals in the AcDex, which was easily controlled with acetalation reaction time. The release was shown in low pH environments, similar to what is found in an infarcted heart. bFGF maintained activity after release from the microparticles. Finally, biocompatibility of the microparticles was assessed.

Controllable dual protein delivery through electrospun fibrous scaffolds with different hydrophilicities.

Tissue engineered scaffolds should actively participate not only in structural support but also in functional tissue regeneration. Thus, novel smart biomaterial scaffolds have been developed, which incorporate a variety of bioactive molecules to accelerate neo-tissue formation. The effective delivery of multiple bioactive molecules with distinct kinetics to target sites at an appropriate concentration and in a timely manner is desired to drive tissue development to completion. To achieve effective, controllable delivery of multiple factors, a dual protein delivery system has been developed by electrospinning poly(lactide-co-glycolide) (PLGA) with different hydrophilicities. Bovine serum albumin or myoglobin was incorporated into and released gradually from these electrospun fibrous PLGA scaffolds. All the scaffolds exhibited similar loading efficiencies of approximately 80% of the target proteins. The introduction of Pluronic F-127 (PF127) dramatically increased scaffold hydrophilicity, which affected the release kinetics of these proteins from the scaffolds. Furthermore, distinct protein release patterns were achieved when using dual protein-loaded scaffolds with different hydrophilicities when these scaffolds were fabricated by co-electrospinning. This system may be useful as a method for delivering multiple bioactive vehicles for tissue engineering applications.

Tailored synthesis of intelligent polymer nanocapsules: an investigation of controlled permeability and pH-dependent degradability.

In this study, we present a new route to synthesize an intelligent polymer nanocapsule with an ultrathin membrane based on surface-initiated reversible addition-fragmentation chain-transfer polymerization. The key concept of our report is to use pH-responsive polydiethylaminoethylmethacrylate as a main membrane-generating component and a degradable disulfide bond to cross-link the membrane. The permeability of membrane, tuned by adjusting pH and using different lengths of the cross-linkers, was proven by showing a dramatic swelling behavior of the nanocapsules with the longest cross-linker from 560 nm at pH 8.0 to 780 nm at pH 4.0. Also, due to the disulfide cross-linker, degradation of the capsules using GSH as reducing agent was achieved which is further significantly promoted at pH 4.0. Using a rather long-chain dithiol cross-linker, the synthesized nanocapsules demonstrated a good permeability allowing that an enzyme myoglobin can be postencapsulated, where the pH controlled enzyme activity by switching membrane permeability was also shown.

Controlled-release and preserved bioactivity of proteins from (self-assembled) core-shell double-walled microspheres.

In order to address preserved protein bioactivities and protein sustained-release problems, a method for preparing double-walled microspheres with a core (protein-loaded nanoparticles with a polymer-suspended granule system-formed core) and a second shell (a polymer-formed shell) for controlled drug release and preserved protein bioactivities has been developed using (solid-in-oil phase-in-hydrophilic oil-in-water (S/O/O(h)/W)) phases. The method, based on our previous microsphere preparation method (solid-in-oil phase-in-hydrophilic oil-in-water (S/O/O(h)/W), employs different concentric poly(D,L-lactide-co-glycolide), poly(D,L-lactide), and protein-loaded nanoparticles to produce a suspended liquid which then self-assembles to form shell-core microspheres in the hydrophilic oil phase, which are then solidified in the water phase. Variations in the preparation parameters allowed complete encapsulation by the shell phase, including the efficient formation of a poly(D,L-lactide) shell encapsulating a protein-loaded nanoparticle-based poly(D,L-lactide-co-glycolide) core. This method produces core-shell double-walled microspheres that show controlled protein release and preserved protein bioactivities for 60 days. Based upon these results, we concluded that the core-shell double-walled microspheres might be applied for tissue engineering and therapy for chronic diseases, etc.

Protein diffusion in photopolymerized poly(ethylene glycol) hydrogel networks.

In this study, protein diffusion through swollen hydrogel networks prepared from end-linked poly(ethylene glycol)-diacrylate (PEG-DA) was investigated. Hydrogels were prepared via photopolymerization from PEG-DA macromonomer solutions of two molecular weights, 4600 Da and 8000 Da, with three initial solid contents: 20, 33 and 50 wt/wt% PEG. Diffusion coefficients for myoglobin traveling across the hydrogel membrane were determined for all PEG network compositions. The diffusion coefficient depended on PEG molecular weight and initial solid content, with the slowest diffusion occurring through lower molecular weight, high-solid-content networks (D(gel) = 0.16 ± 0.02 × 10(-8) cm(2) s(-1)) and the fastest diffusion occurring through higher molecular weight, low-solid-content networks (D(gel) = 11.05 ± 0.43 × 10(-8) cm(2) s(-1)). Myoglobin diffusion coefficients increased linearly with the increase of water content within the hydrogels. The permeability of three larger model proteins (horseradish peroxidase, bovine serum albumin and immunoglobulin G) through PEG(8000) hydrogel membranes was also examined, with the observation that globular molecules as large as 10.7 nm in hydrodynamic diameter can diffuse through the PEG network. Protein diffusion coefficients within the PEG hydrogels ranged from one to two orders of magnitude lower than the diffusion coefficients in free water. Network defects were determined to be a significant contributing factor to the observed protein diffusion.

Anionic and cationic dextran hydrogels for post-loading and release of proteins.

In this study, post-loading of proteins in and release from chemically crosslinked dextran hydrogels exploiting reversible electrostatic interactions was investigated. Methacrylated dextran (Dex-MA) was co-polymerized with methacrylic acid (MA) or dimethylaminoethyl methacrylate (DMAEMA) to form negatively and positively charged hydrogels, respectively. Incubation of negatively charged hydrogels in a low ionic strength (10 mM HEPES, pH 7.4) solution of cytochrome C (isoelectric point (pI) 10.2) led to quantitative absorption of the protein in the hydrogel. BSA (pI 4.8) and myoglobin (pI 7.2) were post-loaded into positively charged gels at neutral pH and negatively charged gels at pH 5, respectively. Loading efficiency and protein distribution in the gels were dependent on network charge (maximum loading efficiency at 100-150 µmol charged monomer/g gel) and crosslink density (higher and more homogenous loading at lower crosslink density) and on the ionic strength during loading (lower but more homogenous loading at higher ionic strength). Diffusion controlled release of the loaded protein was triggered by incubation of the hydrogel in HEPES buffered saline (HBS) pH 7.4. The amount of released cytochrome C in HBS varied from 94% to 70% from gels containing 60 and 150 MA/g, respectively. Importantly, quantitative release was obtained in 1 M NaCl, indicating that post-loading led to neither the formation of insoluble protein aggregates nor irreversible immobilization of the protein in the matrix. ESI-MS analysis of the released cytochrome C revealed that post-loading did not result in oxidation of the protein, as opposed to loading during preparation of the gels. In conclusion, this paper shows that post-loading of proteins in dextran hydrogels and release exploiting reversible charge interactions can be applied for efficient loading of proteins that are negative, positive or neutral at physiological pH. Importantly, our data demonstrate that using this loading method no chemical modification to the protein occurred.

Design of recombinant hemoglobins for use in transfusion fluids.

Molecular biology has been applied to the development of hemoglobin-based oxygen carrier (HBOC) proteins that can be expressed in bacteria or yeast. The transformation of the hemoglobin molecule into an HBOC requires a variety of modifications for rendering the acellular molecule of hemoglobin physiologically acceptable when transfused in circulation. Hemoglobins with different oxygen affinities can be obtained by introducing mutations at the heme pocket, the site of oxygen binding, or by introducing surface mutations that stabilize the hemoglobin molecule in the low-oxygen-affinity state. Modification of the size of the heme pocket is also used to hinder nitric oxide depletion and associated vasoconstriction. Introduction of cysteine residues on the hemoglobin surface allows formation of intermolecular bonds and formation of polymeric HBOCs. These polymers of recombinant hemoglobin have the characteristics of molecular size, molecular stability, and oxygen delivery to hypoxic tissue suitable for an HBOC.

Development of protein delivery microsphere system by a novel S/O/O/W multi-emulsion.

A novel method has been developed to protect protein drugs in poly (lactic-co-glycolic acid) (PLGA) microspheres using S/O/O/W multi-emulsion method. This method develops a novel protein drug sustained-release system, which is based on the combination of protein-loaded dextran glassy microparticles (protein-loaded AqueSpheres) and PLGA microspheres. The protein molecules are encapsulated in the dextran glassy particles and the drug-containing dextran glassy particles are further dispersed in the PLGA microspheres. The protein-loaded AqueSpheres based PLGA composite microspheres have spherical shape and a smooth surface. They possess a normal size distribution and a mean diameter of 67.08 microm. The method may decrease protein aggregations and incomplete release due to avoiding protein contacting with oil/water interfaces and hydrophobic PLGA. The dextran glassy particles can stabilize proteins in the PLGA matrix, which is the major advantage of this novel protein sustained-release system over preparation for the PLGA microspheres using W/O/W double-emulsion method.

Biodegradable nanoparticles of partially methylated fungal poly(beta-L-malic acid) as a novel protein delivery carrier.

The preparation of nanoparticles from 75% methylated poly(beta-L-malic acid) is described. Their degradation in aqueous environments was examined and the influence of pH and lipase on the rate of hydrolysis was evaluated. Six proteins were used to estimate the loading efficiency of the nanoparticles. The amount of protein retained in the nanoparticles was found to depend on the acid/basic character of the protein. Protein release from the loaded nanoparticles upon incubation in water under physiological conditions encompassed polymer hydrolysis and happened steadily within 3-10 d. The activity loss of entrapped alpha-chymotrypsin caused by loading and releasing depended on the method used for loading.

5-methyl-pyrrolidinone chitosan films as carriers for buccal administration of proteins.

The purpose of this research was to investigate 5-methyl-pyrrolidinone chitosan (MPC) films as carriers for buccal delivery of protein drugs. Placebo and protein-loaded MPC films were prepared by casting and were then cross-linked with tripolyphosphate at different pH conditions. Myoglobin (MHb) was chosen as the model protein because its molecular weight is under the permeability limit of the buccal mucosa. The observed characteristics like bioadhesiveness, swelling behavior, and in vitro release of MHb from loaded films furnish information on the functional behavior of these films. The results obtained show that the modulation of MHb release was achieved only through chitosan cross-linking; the best results in release rate control were obtained by cross-linking performed at pH 6.5. Good bioadhesion properties were maintained even with high cross-linking degrees; the swelling index of MHb-loaded films at different cross-linking degrees evaluated at pH 7.4 and pH 6.4 were comparable to those of placebo films. By setting suitable tripolyphosphate cross-linking conditions for MPC films, one can control protein release without affecting bioadhesion.

Lipid hydroperoxidase activity of myoglobin and phenolic antioxidants in simulated gastric fluid.

Our recent study demonstrated the potential of gastric fluid at pH 3.0 to accelerate lipid peroxidation and cooxidation of dietary constituents in the stomach medium. Metmyoglobin is known to catalyze the breakdown of lipid hydroperoxides to free radicals, a reaction that could enhance the propagation step and general lipid peroxidation. During this reaction, a part of the free radicals is autoreduced by metmyoglobin. At pH 3.0, metmyoglobin at low concentration was almost 7 x 10(4) times as effective as at pH 7.0 in enhancing the rate of lipid peroxidation. Our study demonstrated that metmyoglobin, at a low concentration (approximately 1:30), as compared with that of the hydroperoxides in the lipid system, worked prooxidatively increasing the amounts of linoleate hydroperoxides. However, at a high concentration (approximately 1:3), metmyoglobin acted antioxidatively and decomposed hydroperoxides, whose concentrations then remained at zero for a long time. Catechin, a known polyphenol, supports the inversion of metmyoglobin catalysis, from prooxidation to antioxidation. The antioxidative activity of the couple metmyoglobin-catechin was better at pH 3.0 than at pH 7.0, indicating that this reaction is more dependent on metmyoglobin than on catechin. During this reaction, catechin or quercetin not only donates reducing equivalents to prevent lipid peroxidation but also prevents the destruction and polymerization of metmyoglobin. The results of this research highlighted the important and possible reactions of heme proteins and polyphenols as couple antioxidants, working as hydroperoxidases or as pseudo-peroxidases. We hypothesize that the occurrence of these reactions in the stomach could have an important impact on our health and might help to better explain the health benefits of including foods rich in polyphenol antioxidants in the meal, especially when consuming red meat.

Concentration effects in myoglobin-catalyzed peroxidation of linoleate.

The concentration of the free fatty acid anion linoleate was found to be important for the pro-oxidative activity of metmyoglobin, MbFe(III), and for mixtures of metmyoglobin and hydrogen peroxide, MbFe(III)/H(2)O(2), to yield perferrylmyoglobin, (*)MbFe(IV)=O, whereas for ferrylmyoglobin, MbFe(IV)=O, no concentration effect was noted as studied in linoleate emulsions (pH 7.4 and 25 degrees C). Determination of conjugated dienes using second-derivative absorption spectroscopy, changes in Soret band absorbance, and spin-trapping ESR spectroscopy with alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) as the spin trap were used to evaluate the pro-oxidative activity of myoglobins. At a linoleate (LA)/heme protein (HP) ratio of 100, no MbFe(III)-induced linoleate peroxidation was observed, as MbFe(III) was converted to its non-pro-oxidative low-spin derivative, hemichrome, independently of the presence of H(2)O(2). At higher LA/HP ratios, linoleate peroxidation was initiated by the addition of MbFe(III), both in the presence and in the absence of H(2)O(2). This proceeded with denaturation of MbFe(III), as followed by changes in Soret absorption band, which most probably release or expose the heme group to the environment and thereby permit hematin-induced lipid peroxidation. The obtained results show that the mechanism by which MbFe(IV)=O initiates linoleate peroxidation is different from MbFe(III)- and MbFe(III)/H(2)O(2)-initiated linoleate peroxidation. The shift in mechanism between heme protein cleavage of lipid hydroperoxides and hematin-induced lipid peroxidation is discussed in relation to oxidative progress in biological systems and muscle-based foods.

Preparation and release characteristics of protein-loaded polyanion/gelatin complex.

PURPOSE: This paper describes preparation of polymethacrylic acid/gelatin complex and Myoglobin release characteristics in order to evaluate the polyanion/gelatin complexes as matrices that can release proteins at a near zero-order kinetics over a long period of time. METHODS: Mb-loaded PMAA/gelatin complex was prepared by two different titration methods. Mb entrapment efficiency and PMAA/gelatin ratio in the complex were determined by HPLC. The release of Mb and gelatin from the complex was followed by HPLC. Mb conformation was detected by UV-vis spectrophotometer and capillary electrophoresis apparatus. RESULTS: Polyanion/gelatin feed ratio of the polyanion/gelatin/Mb mixed solution has great effects on complex yield and protein entrapment efficiency when "Type I" titration method is adopted, while for the colloid titration method the complex yield and protein entrapment efficiency are hardly influenced by preparative conditions (ca. 100%). Mb release rate could be adjusted by the complex composition (e.g., PMAA MW, hydrophobilization of PMAA, Mb loading and PMAA/gelatin ratio, etc.). Moreover, by coating of high MW PMAA/gelatin complex cylinder in a hydrophobic membrane with one open-end left, the period of protein release can extend to ca. 20 days and the release displays a near zero-order pattern. The protein release profiles can be described by the dissociation/erosion mechanism. The entrapment process has little effect on Mb conformation. CONCLUSIONS: The studied polyanion/gelatin complex is promising to be used as protein carriers to release proteins at a near zero-order kinetics over a long period of time by selecting suitable polyanions and designing the device structure.

Pulsatile protein release from a laminated device comprising polyanhydrides and pH-sensitive complexes.

A laminated device comprising of polyanhydrides as isolating layers and pH-sensitive complexes as protein-loaded layers was designed to deliver proteins in a pulsatile manner. Poly(sebacic anhydride)-b-polyethylene glycol (PSA-b-PEG) and poly(trimellitylimidoglycine-co-sebacic anhydride)-b-polyethylene glycol (P(TMA-gly-co-SA)-b-PEG) were synthesized as isolating layers for their good processing properties at room temperature and suitable erosion duration. During the erosion period, pH of the dissolution fluid decreases to a low value (3.8-5.8). Poly(methacrylic acid)/polyethoxazoline (PMAA/PEOx) complex was used as protein-loaded layers, which could dissociate and release model proteins, Myoglobin (Mb) and Bovine Serum Albumin (BSA), at pH 7.4 while become stable and retained the drugs below pH 5.0. The protein release from the device showed a typical pulsatile fashion. The lag time prior to the pulsatile protein release correlated with the hydrolytic duration of the polyanhydrides, which varied from 30 to 165 h by selecting polyanhydride type and isolating layer thickness. In addition, the pulse duration could be adjusted from 18.5 to 40 h by varying the mass of the complex. The results can be attributed to the synergistic effects between the degrading polyanhydrides, pH-sensitive complexes and proteins.

Myoglobin clearance during continuous veno-venous hemofiltration with or without dialysis.

The management of acute myoglobinuric renal failure, the major complication of rhabdomyolysis, continues to be a treatment dilemma for the clinician as limited therapeutic options are available. Previously, we have demonstrated that continuous arteriovenous hemofiltration (CAVH) is an effective technique for removing myoglobin in an animal model. In the present study, swine were administered four grams of equine myoglobin intravenously and underwent the continuous veno-venous hemofiltration (CVVH) procedure for six hours each. Animals were studied in each of the following groups: CVVH at a pump rate 100 ml/minute, CVVH at a pump rate 200 ml/minute and CVVH at a pump rate 100 ml/minute plus dialysis at a dialysate flow rate of one Liter/h. Once the filtering process was initiated there was a rapid and sustained production of ultrafiltrate in all groups. The amount of myoglobin excreted in the ultrafiltrate over the six-hour filtering period was 688, 948 and 570 mg which corresponded to 17, 24 and 14 percent of the administered dose, respectively, for the three treatments. In comparison to previous CAVH experiments, CVVH removed more circulating myoglobin and the addition of the dialysis component did not appear to improve removal. Based on these findings, it appears that the CVVH hemofiltration system is a viable option for the removal of systemic myoglobin.

Following antigen challenge, T cells up-regulate cell surface expression of CD4 in vitro and in vivo.

The low precursor frequency of Ag-specific T cells has raised significant barriers to studying the T cell response in vivo. We demonstrate that T cells up-regulate the cell surface expression of CD4 following Ag recognition, which identifies Ag-specific T cells in vitro and in vivo and allows their characterization. The CD4high cell subpopulation contains the Ag-specific population as indicated by Ag-induced proliferation and limiting dilution analyses. The use of the CD4high marker will allow analysis of the dynamics of the T cell immune response in vivo, the study of the suboptimal T cell response to Ag, and the identification of T cells which are reactive to known and unknown autoantigens.