Synthesis of pH and Glucose Responsive Silk Fibroin Hydrogels

Silk fibroin (SF) has attracted much attention due to its high, tunable mechanical strength and excellent biocompatibility. Imparting the ability to respond to external stimuli can further enhance its scope of application. In order to imbue stimuli-responsive behavior in silk fibroin, we propose a new conjugated material, namely cationic SF (CSF) obtained by chemical modification of silk fibroin with ε-Poly-(L-lysine) (ε-PLL). This pH-responsive CSF hydrogel was prepared by enzymatic crosslinking using horseradish peroxidase and H₂O₂. Zeta potential measurements and SDS-PAGE gel electrophoresis show successful synthesis, with an increase in isoelectric point from 4.1 to 8.6. Fourier transform infrared (FTIR) and X-ray diffraction (XRD) results show that the modification does not affect the crystalline structure of SF. Most importantly, the synthesized CSF hydrogel has an excellent pH response. At 10 wt.% ε-PLL, a significant change in swelling with pH is observed. We further demonstrate that the hydrogel can be glucose-responsive by the addition of glucose oxidase (GOx). At high glucose concentration (400 mg/dL), the swelling of CSF/GOx hydrogel is as high as 345 ± 16%, while swelling in 200 mg/dL, 100 mg/dL and 0 mg/dL glucose solutions is 237 ± 12%, 163 ± 12% and 98 ± 15%, respectively. This shows the responsive swelling of CSF/GOx hydrogels to glucose, thus providing sufficient conditions for rapid drug release. Together with the versatility and biological properties of fibroin, such stimuli-responsive silk hydrogels have great potential in intelligent drug delivery, as soft matter substrates for enzymatic reactions and in other biomedical applications.     

Egg phosphatidylcholine and dioleoylphosphatidylethanolamine liposomes containing acid proteinoid: Comparison of pH‐sensitivity

pH‐Sensitive liposomes were prepared by modifying the surfaces of egg phosphatidylcholine (EPC) liposomes and dioleoylphosphatidylethanolamine (DOPE) liposomes with an acidic proteinoid. The acidic proteinoid (Prot AL) was prepared by a melt‐condensation of aspartic acid and leucine (98.5:1.5 mol/mol). The maximum amount of Prot AL accommodated by EPC liposomes without loss of the fluorescence quenching of calcein occurred when the ratio of Prot AL to EPC was 1:2. The EPC liposomes exhibited pH‐dependent release but the degree of release in 5 min was less than 10% in the range of pH 6.0–8.0. A marked increase in release was observed at pH 5.5 and the degree of release was about 38%. Acidification‐induced contraction of Prot AL may impose a mechanical stress on the liposomal membrane, deforming and demaging the membrane. On the other hand, a high fluorescence quenching, more than 60%, was obtained when the ratio of Prot AL to DOPE was 5.5:10. The pH‐sensitivity of DOPE liposomes bearing the proteinoid was much higher than that of egg PC liposomes bearing the same proteinoid. Following the changes in the size with varying pH, DOPE liposomes seemed to be disintegrated.     

Extraction of cadmium from solutions containing various heavy metal ions by Amberlite LA-2

In present study, selective extraction of cadmium from acidic leach solutions, containing various heavy metal ions, by emulsion liquid membrane (ELM) is studied. For this reason, the zinc plant copper cake was leached with sulfuric acid and main acidic leach solution containing Zn(II), Cu(II), Fe(II), Cd(II), Co(II) and Ni(II) ions was obtained. After Zn(II), Cu(II), Fe(II) and Cd(II) ions in the acidic leach solution were separated, the important parameters influencing the extent of cadmium extraction were investigated and optimum conditions were determined. Cadmium extraction was influenced by number of parameters like initial metal ion concentration, mixing speed, phase ratio, extractant concentration, surfactant concentration, the stripping solution type and concentration, and the feed solution acid concentration. The optimum values of parameter above mentioned were used and cadmium in the acidic leach solution containing 650 mg Cd/L, 365 mg Co/L, 535 mg Ni/L, and 1260 mg Zn/L was almost completely extracted within 10 min. The results showed that it is possible to extract 99% of cadmium after 10 min contact time by using ELM from aqueous solutions, containing Fe(II), Al(III), Cu(II), Zn(II), Pb(II), Co(II) and Ni(II) ions, at the optimum operating conditions.     

An improved assay for cholesterol 7α-hydroxylase activity using phospholipid liposome solubilized substrate

A persistent problem in measurement of cholesterol 7α-hydroxylase (7α-OHase) activity by isotope incorporation has been solubilization of cholesterol substrate. Solubilization with Tween 20, for example, resulted in a 75% reduction in 7α-OHase activity after a 60 min incubation of substrate with microsomes. Incorporation of cholesterol substrate into small, unilamellar phospholipid vesicles (liposomes) prevented this effect, resulting in a 50% increase in activity over the same 60 min incubation at optimal concentrations. Using cholesterol in liposomes as substrate, standard assay conditions were determined to be: preparation of liposomes with 180 μM cholesterol substrate and 0.5 mg phospholipid/assay; incubation of these liposomes with 0.5 mg microsomal protein at 37 C for 60 min; addition of a NADPH generating system to start the reaction, and incubation at 37 C for 30 min before stopping the reaction and determining the amount of 7α-hydroxycholesterol formed. In addition to preventing the detergent-related inhibition of the enzyme, liposome-solubilized substrate also reduced the variation among replicates from a coefficient of 45% with Tween 20 to 4.2% with phospholipid. This method provides a sensitive and reliable alternative to methods which require more sophisticated equipment and allows total control of substrate concentration in a form readily accessible to the enzyme. This work was reported in part at the American Society of Biological Chemists Meeting, St. Louis, Missouri, June 1984.     

Effect of <Emphasis Type="Italic">Fomitopsis pinicola</Emphasis> extract on blood glucose and lipid metabolism in diabetic rats

This study was performed to investigate the effects of Fomitopsis pinicola extract on blood glucose and lipid metabolism in diabetic rats. The blood glucose concentration was similar to that of the control at 30 min, but after 60 min of glucose administration the blood glucose concentration rapidly decreased, and after 120 min was 100.7±4.0 mg/dL, representing an approximate 50% decrease compared to the control. In the case of the diabetic rats induced by streptozotocin, the concentration of blood glucose was decreased from 362.0±16.7 to 204.5±11.4 mg/dL after 20 days of administration. HDL- and LDL-cholesterol concentrations were 39.0±4.3 mg/L and 13.2±3.4 mg/dL, respectively, representing an approximate increase of 73% and approximate decrease of 76%, respectively, compared to the control. The activities of aspartate aminotransferase and alanine transaminase were increased. On the other hand, activities of amylase, alkaline phosphatase, lactate dehydrogenase, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase were decreased compared to that of the control. No difference was evident between test and control rats with respect to white blood cell, red blood cell, hemocyte, hemoglobin, hematocrit, and platelet counts. These results indicate that F. pinicola extract is useful as a preventative and treatment agent for damage of liver and kidney cells.     

Catalytical conversion of fructose and glucose into 5-hydroxymethylfurfural in hot compressed water by microwave heating

Production of 5-hydroxymethylfurfural (5-HMF) from glucose and fructose catalyzed by TiO₂ and ZrO₂ under microwave irradiation was studied. For the case of TiO₂ used in the fructose reaction, 5-HMF yield was 38.1% for a fructose conversion of 83.6% for 5 min reaction time. A 5-HMF yield of 30.5% for a fructose conversion of 65% was obtained for 5 min reaction time in the presence of ZrO₂. The ZrO₂ was found to promote isomerization of glucose to fructose, in which the selectivity of fructose from glucose became higher than 60% for about 50% glucose conversion for a reaction time of 1 min. Under the conditions (5 ml of 2 wt% fructose solution, 0.05 g of TiO₂, 200 °C, and 3 min), fructose conversion and HMF yields by microwave heating (73% and 35%, respectively) were higher than those by sand bath heating (27% and 12%, respectively).     

The selective delivery of anticancer agents using target-sensitive liposomes

Target-sensitive (TG-S) liposomes having modified antibodies on their surface were employed to study the release of calcein and the selective delivery of the anticancer agents, doxorubicin (DOX) and methotrexate (MTX). The release of calcein from TG-S liposome occurred when the various target cells were contacted with liposomes and it was proportionally increased with the increase of antibody affinity to the target cells. Increasing the concentration of antigen molecules (major histocompatibility, MHC) on the surface of RMA-S, the release of calcein and drugs from TG-S liposomes contacting with RMA-S also rised. The destabilization of TG-S liposomes was only induced above a threshold density of surface antigen on the target cell membrane. The growth inhibition of specific target cells by the liposomal drugs was always stronger than that of the non-specific ones. For specific target cells, the IC₅₀ of liposomal DOX was about 2 times greater than that of free DOX, on the while, for non-specific target cells, more than 5 times. This indicates that the liposomal drugs were transferred preferentially to the specific target cells than the non-specific ones. Based on this phenomenon, the TG-S liposomal MTX were also applied for the selective elimination of the specific target cells in the mixed culture of specific and non-specific target cells.     

Synthesis and study of copolymer of vinylferrocene,acrylamide and 2-(diethylamino)ethyl methacrylate as a polymeric mediator for electrochemical biosensors

Copolymer of vinylferrocene, acrylamide and 2-(diethylamino)ethyl methacrylate was synthesized by free-radical polymerization. Proton nuclear magnetic resonance and inductively coupled plasma-optical emission spectroscopy confirmed successful incorporation of ferrocene moieties in the copolymer. The effect of monomer feed ratio on copolymer molecular weight was studied and it was found that the increase in 2-(diethylamino)ethyl methacrylate fraction in the monomer feed facilitated incorporation of vinylferrocene and led to decrease in copolymer molecular weight. The synthetic process was scaled up in a pilot plant and an ultrafiltration method was developed to reduce residual levels of each monomer below 0.1% w/w. The copolymer displayed reversible redox electrochemical characteristics of the ferrocene moieties and mediation of glucose oxidation with enzyme flavin adenine dinucleotide-glucose dehydrogenase in phosphate buffered saline. A diffusion coefficient of 3.36 × 10^?7 cm²/s for the dissolved polymeric mediator was estimated from Randles–Sevcik analysis. The copolymer and the enzyme were mixed with water-based carbon ink and then screen-printed to form working electrodes. These biosensors established equilibrium current responses in less than 3 min following step changes of glucose concentration within the clinically relevant range of 8–735 mg/dL when exposed to a continuous flow of blood at 15 μL/min. The calibration plot of three biosensors aligned closely to a quadratic regression line (R square 0.9909) suggesting reasonable sensor measurement reproducibility and low levels of ferrocene leaching out of the polymeric mediator during the 7.5 h test period. A Michaelis constant for the sensor was estimated at 655 mg/dL.     

Radiation synthesis of graphene oxide/composite hydrogels and their ability for potential dye adsorption from wastewater

A high yield of graphene oxide (GO) was chemically synthesized from graphite powder utilizing adjusted Hummer's method. The contents of acidic functional groups in GO were determined using potentiometric titration. Composite hydrogels dependent on graphene oxide/poly(2-acrylamido-2-methylpropanesulfonic acid)/polyvinyl alcohol (GO/PAMPS/PVA) were synthesized utilizing a ⁶⁰Co gamma irradiation source at different doses. The synthesized graphene oxide and composite hydrogels were portrayed via X-ray diffraction, thermogravimetric analysis, and Fourier transform infrared analysis. The morphology of composite hydrogels was characterized by scanning electron microscope. The gel % and swelling % for the prepared hydrogel demonstrated that the swelling % of hydrogel increased with raising AMPS content. Whereas the increment of GO and increasing the irradiation dose lead to a reduction in the swelling %. The influences of pH, GO percentage, initial dye concentration, the adsorbent dosage, contact time, and temperature on the adsorption of basic blue 3 dye were evaluated and the adsorption capacity was 194.6 mg/g at optimum conditions; pH = 6, GO/PAMPS/PVA composite hydrogels with 5 wt% of GO, initial dye concentration = 200 mg/L, adsorbent dose = 0.1 g, solution volume = 50 mL after 360 min at room temperature (25°C). The adsorption of dye onto the GO/PAMPS/PVA composite hydrogels follows Pseudo-second-order adsorption kinetics, fits the Freundlich adsorption isotherm model.     

Direct Coagulation Casting of Alumina Suspension from Calcium Citrate Assisted by pH Shift

Direct coagulation casting of alumina suspension via controlled release of high valence counter ions (DCC–HVCI) using calcium citrate as coagulating agent was reported. Hydrolysis of glycerol diacetate shifts the pH of suspension to weakly acidic region which helps to decompose calcium citrate and release calcium ions. The effect of concentration of glycerol diacetate and calcium citrate on the pH and viscosity of alumina suspension was investigated at 25°C and 60°C, respectively. The pH of suspensions with glycerol diacetate and calcium citrate decreases to 8.6 and 7.5 treated at 25°C and 60°C, respectively. It is indicated that high viscosity is achieved by adding 2 vol% glycerol diacetate and 0.5 wt% calcium citrate which is enough to coagulate the suspension. Green body with compressive strength of 1.0 MPa is obtained by treating the alumina suspension with 2 vol% glycerol diacetate and 0.5 wt% calcium citrate at 60°C for 1 h. The alumina ceramics sintered at 1550°C have homogeneous microstructure with relative density above 99.0%.     

Remediation of soil contaminated with lubricating oil by extraction using subcritical water

The remediation of lubricating oil contaminated soil was investigated by extraction using subcritical water. The effects of temperature and time on extraction efficiency were studied by performing eight individual extractions and varying the subcritical water temperature (200, 225, 250, and 275 °C) and extraction time (90, 120, 180, and 240 min) in a dynamic mode. Also, a comparison was carried out of the feasibility of two operational modes, namely, dynamic and static-dynamic mode. Of the 25,088 mg/kg of lubricating oil as the total petroleum hydrocarbon (TPH) concentration in untreated soil, the residual concentration was found to be ∼500 and 235 mg/kg for after 120 min extraction in a lab-scale apparatus and 150 min extraction in a 30-fold scale-up experiment, respectively, at 275 °C in static-dynamic mode. The result of this study showed the significant effect of the static-dynamic mode on extraction efficiency. The time and volume of water needed for the static-dynamic mode were much lower than those needed for the dynamic mode. These results are of practical interest in developing the subcritical water extraction technology for extraction of lubricating oil and, in a broad sense, petroleum hydrocarbons contaminated soil.     

Direct Coagulation Casting of Alumina Suspension via Controlled Release of High Valence Counterions from Thermo‐sensitive Liposomes

Thermo‐sensitive liposomes were prepared using reverse phase evaporation method using natural lipid egg phosphatidylcholine (EPC) and cholesterol (CH). Inorganic salts containing high valence counterions (HVCI) are encapsulated by the liposomes. The phase transition temperature of the liposome is at 38°C with 50 wt% addition of cholesterol. The encapsulation rate of liposomes reaches 85% for high valence anion (SO₄^2?) and 55% for high valence cation (Ca²⁺). The liposomes are introduced into ceramic colloidal forming and dispersed in the suspension for identical charge with alumina particles at room temperature. The release of HCVI from the liposomes can coagulate the alumina suspension after heating at 38°C for 3 h, but the de‐moldable time is ~ 6–7 h. Dense ceramic products with relative density of above 98% and uniform microstructure can be prepared by this method without burnout process.     

Conformational Changes of Anoplin,W-MreB1–9, and (KFF)3K Peptides near the Membranes

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB_1–9, and one cell-penetrating peptide, (KFF)₃K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB_1–9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB_1–9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.     

Advances in the control of electrophoretic process parameters to tune the ytterbium disilicate coatings microstructure

Suspensions of ytterbium disilicate in isopropanol were prepared using iodine dispersant. Their zeta potential, electrical conductivity, and pH dependence with iodine concentration is detailed. Electrophoretic deposition was performed on silicon substrates at various voltages (100-200 V) and times (until 10 minutes) and the growth dynamic was investigated. It was observed that the deposited mass reaches a maximum value for [I₂] = 0.2 g/L, and the coating microstructure becomes porous at higher iodine concentrations. Current density and voltage measurements allowed to correlate this behavior to the increase of free protons concentration in the suspension. In these conditions, it was proved that porosity increases with the increase in applied voltage, and a compaction occurs as the deposition time increases. This has been related to the coating resistance increase and subsequent decrease in effective voltage in the suspension. The denser coatings (20% of porosity) were obtained in the case of suspension without iodine, at the minimum applied voltage and for the longest deposition times.     

Optimization on condition of epigallocatechin-3-gallate (EGCG) nanoliposomes by response surface methodology and cellular uptake studies in Caco-2 cells

The major component in green tea polyphenols, epigallocatechin-3-gallate (EGCG), has been demonstrated to prevent carcinogenesis. To improve the effectiveness of EGCG, liposomes were used as a carrier in this study. Reverse-phase evaporation method besides response surface methodology is a simple, rapid, and beneficial approach for liposome preparation and optimization. The optimal preparation conditions were as follows: phosphatidylcholine-to-cholesterol ratio of 4.00, EGCG concentration of 4.88 mg/mL, Tween 80 concentration of 1.08 mg/mL, and rotary evaporation temperature of 34.51°C. Under these conditions, the experimental encapsulation efficiency and size of EGCG nanoliposomes were 85.79% ± 1.65% and 180 nm ± 4 nm, which were close with the predicted value. The malondialdehyde value and the release test in vitro indicated that the prepared EGCG nanoliposomes were stable and suitable for more widespread application. Furthermore, compared with free EGCG, encapsulation of EGCG enhanced its inhibitory effect on tumor cell viability at higher concentrations.     

Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression

Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.     

Residence time distribution analysis and optimization of photocatalysis of phenazopyridine using immobilized TiO2 nanoparticles in a rectangular photoreactor

Optimization of photocatalytic degradation of phenazopyridine (PhP) under UV light irradiation using immobilized TiO₂ nanoparticles was studied. The effect of operational parameters was investigated using response surface methodology (RSM). Maximum removal efficiency was achieved at the optimum conditions: initial drug concentration of 10 mg/L, UV light intensity of 47 W/m², flow rate of 200 mL/min, and reaction time of 150 min. The residence time distribution (RTD) analysis was studied to find the effect of flow rate on the drug removal efficiency. The tracer (PhP) pulse injection response was studied with UV–vis measurements and was used to prepare RTD curves.     

Kinetics,aggregation behavior and optimization of the fractionation of whey protein isolate with hydrochloric acid

Concentrated WPI solutions (10%, w/w) containing approximately 50% beta-lactoglobulin (β-LG) and 26% alpha-lactalbumin (α-LA) were fractionated with HCl at acidic pH and moderate temperature to obtain enriched α-LA and β-LG fractions. Aggregation behavior and kinetics of protein precipitation and aggregate formation were analyzed as a function of four process parameters: pH (3.0–5.5), temperature (50–70 °C), reaction time (0–180 min) and protein concentration (10–29%). The precipitation and aggregation of α-LA appeared rate-limited, with a logarithmic dependence of time and possible bimodal nucleation rate, and varied considerably with pH and temperature. Aggregates as large as ～300 μm were noted after 120 min at pH 4, 60 °C. Processing parameters were optimized to obtain both a high aggregate yield and optimal composition of the aggregate fraction. The optimally enriched solid and liquid fractions contained 58% α-LA and 76% β-LG, respectively, with 99% and 74% recovery ratios. Over the pH range studied, β-LG aggregation was found negligible at 60 °C and β-LG recovery in the aggregates attributed to liquid holding. Increasing WPI concentration accelerated α-LA aggregation, demonstrating a concentration-dependent aggregation mechanism, and reduced aggregate purity. Enriched whey protein fractions are valuable health-enhancing food ingredients.     

“类乒乓球”状壳聚糖微球对环丙沙星的控制释放性能的研究

以甲醛作为交联剂,通过悬浮交联法得到单分散性的微米级微球。采用分光光度法研究了壳聚糖微球对环丙沙星的载药释药性能,考察了环丙沙星初始浓度、pH、微球粒径大小、载药时间及温度对饱和吸附量的影响。结果表明,在初始浓度为200 mg/L,pH为8.80和时间为65 min,温度为37℃的优化条件下,壳聚糖微球对环丙沙星的载药量最大,最大吸附量为325 mg/g。在pH为7.4,温度为37℃的模拟人体肠胃缓冲溶液(NaH2PO4/NaOH)中研究了初始浓度以及释放时间对释放结果的影响。实验表明,环丙沙星在担载时与环丙沙星的初始浓度有关,浓度越大,担载量越大,但是担载效率和浓度之间无确定的线性关系。在环丙沙星释放初期有明显的释放现象,但是随着时间的推移,药物释放逐渐稳定,释药效率可达97%左右。     

Photo-responsive liposomes decorated with hydrophobically modified poly(vinyl alcohol)–coumarin conjugate

Photo-responsive liposome was developed by modifying the surface of egg yolk phosphatidylcholine (egg PC) liposomes with hydrophobically modified poly(vinyl alcohol)–epoxypropoxy coumarin conjugate (HmPVA-EPC). Decanoyl chloride (DC) was used as a hydrophobic pendant for the hydrophobic modification of PVA. The fluorescence quenching of liposomes was more than 65% when the ratio of lipid/HmPVA-EPC was 1:0.01–1:0.1, but the value was less than 35% in the ratio range of 1:0.2–1:1. Under UV irradiation (λ = 254 nm), egg PC liposomes of which lipid/HmPVA-EPC ratio was 1:0.1 readily release their content for 60 min possibly due to the photo-dimerization of EPC residues.