The Influence of Slag Content on the Structure and Properties of the Interfacial Transition Zone of Ceramisite Lightweight Aggregate Concrete.

Ceramisite lightweight concrete has excellent performance and relatively light self-weight characteristics. At the same time, the recent development of green high-performance concrete and prefabricated components has also brought the abundant utilization of these mineral mixture. An interfacial transition zone exists between the hardened cement paste and the aggregate, which is the weakest part of the concrete, characterized by high porosity and low strength. In order to study the effect of slag content on the interfacial transition zone in lightweight high-strength concrete, experiments were designed to replace cement with slag at different contents (0%, 5%, 10%, 15%). A series of studies was conducted on its macro-strength, microstructure, and composition. The results indicated that the addition of slag improved the porosity and width of the interfacial transition zone. Adding slag did not reduce the thickness of the concrete interfacial transition zone significantly at 3 d, but it led to significant improvement in the thickness of the interfacial transition zone at 28 d, and the thickness of the interfacial zone at 28 d was reduced from 19 µm to 8.5 µm, a reduction of 55%. The minimum value of microhardness in the slurry region of the interfacial specimens also increased from 19 MPa to 26 MPa, an increase of 36%. In addition, the structural density of the interfacial region was further increased, resulting in varying degrees of improvement in the macroscopic anti-splitting strength. One of the important reasons for this phenomenon is that the addition of slag optimizes the chemical composition of the interface and promotes the continuation of the pozzolanic reactivity, which further enhances the hydration at the interface edge.

Structure of Sewage Sludge-Clay Multiscale Composite Particles to Control the Mechanism of SO2 and H2S Gas Release.

In order to address the problem of sulfur gas and other odors released in the process of using sewage sludge as a construction material, this study prepared multiscale composite particles with a "large scale-medium scale-small scale-micro scale" structure by mixing sludge with silica-alumina building materials. Analysis of the structural changes formed by the internal gas of composite particles due to diffusion at different temperatures and a study of the characteristics of SO2 and H2S release from composite particles were conducted, as well as being compared with the release characteristics of pure sludge, which clarified the mechanism of controlling sulfur-containing-gas release from composite particles. The results showed that compared with pure sludge, the sludge-clay multiscale composite particles were able to reduce the release of SO2 and H2S up to 90% and 91%, and the release temperatures of SO2 and H2S were increased to 120 °C and 80 °C, respectively. Meanwhile, the special structure of the sludge-clay multiscale composite particles and the clay composition are the main factors that hinder the diffusion of sludge pyrolysis gases. Additionally, there are three layers of "gray surface layer-black mixed layer-dark gray spherical core" formed inside the composite particles, which is the apparent manifestation of the diffusion of volatile gases. This study provides theoretical support for the application of multiscale composite particle inhibition of odor-release technology in industrial production.

Preparation of Lightweight Ceramsite from Solid Waste Using SiC as a Foaming Agent.

SiC was chosen as the foaming agent, and river bottom silt, waste oil sludge, paint bucket slag, and fly ash were used as raw materials, to prepare lightweight ceramsite without adding any chemical additives. The effects of SiC dosing and sintering temperature on various properties of the ceramsite were studied, and the pore-forming mechanism of the lightweight ceramsite was clarified by thermal analysis and X-ray diffraction analysis. The results showed that the single ceramsite compressive strength, water absorption, bulk density, and porosity of ceramsite sintered at 1180 °C with 1.0% SiC were 2.15 MPa, 2.02%, 490 kg/m3, and 23.85%, respectively. The major mineralogical compositions were quartz, fayalite, and kyanite, with small amounts of albite-low from 1140 to 1190 °C. Furthermore, the concentration of all tested heavy metals from ceramsite was lower than the maximum allowable concentration of the leaching solution specified in the Chinese national standard (GB 5085.3-2007), which reveals that this solid waste ceramsite will not cause secondary environmental pollution. The prepared ceramsite, exhibiting lower bulk density, high water absorption and porosity, and effective solidification of deleterious elements, can be used to prepare green lightweight aggregate concrete. Importantly, preparation of solid waste ceramsite is an effective way to dispose of hazardous wastes.

Influence of Temperature on Characteristics of Particulate Matter and Ecological Risk Assessment of Heavy Metals during Sewage Sludge Pyrolysis.

The formation process of Particulate Matter (PM) during sludge pyrolysis at different temperatures (300-700 °C) and the ecological risks of heavy metals were studied. The results showed that the particulate matter is mainly condensed on the quartz film in a carbon-based organic matter when the pyrolysis temperature was between 200-500 °C in a volatilization process. Inorganic particles was found in the particulate matter when the temperature was raised to 500-700 °C in a decomposition stage. Heavy metals were enriched in particulate matter with increase in pyrolysis temperature. When the temperature reached 700 °C, the concentration of Pb and Cd in the particulate matter significantly increased. The ecological risk assessment showed that heavy metals in the sewage sludge had considerable ecological toxicity. When the pyrolysis temperature reached 700 °C, the ecological toxicity of those heavy metals enriched in the particulate matter decreased considerably.

Experimental Investigation into the Effect of Pyrolysis on Chemical Forms of Heavy Metals in Sewage Sludge Biochar (SSB), with Brief Ecological Risk Assessment.

Experimental investigations were carried out to study the effect of pyrolysis temperature on the characteristics, structure and total heavy metal contents of sewage sludge biochar (SSB). The changes in chemical forms of the heavy metals (Zn, Cu, Cr, Ni, Pb and Cd) caused by pyrolysis were analyzed, and the potential ecological risk of heavy metals in biochar (SSB) was evaluated. The conversion of sewage sludge into biochar by pyrolysis reduced the H/C and O/C ratios considerably, resulting in stronger carbonization and a higher degree of aromatic condensation in biochar. Measurement results showed that the pH and specific surface area of biochar increased as the pyrolysis temperature increased. It was found that elements Zn, Cu, Cr and Ni were enriched and confined in biochar SSB with increasing pyrolysis temperature from 300-700 °C; however, the residual rates of Pb and Cd in biochar SSB decreased significantly when the temperature was increased from 600 °C to 700 °C. Measurement with the BCR sequential extraction method revealed that the pyrolysis of sewage sludge at a suitable temperature transferred its bioavailable/degradable heavy metals into a more stable oxidizable/residual form in biochar SSB. Toxicity of heavy metals in biochar SSB could be reduced about four times if sewage sludge was pyrolyzed at a proper temperature; heavy metals confined in sludge SSB pyrolyzed at about 600 °C could be assessed as being low in ecological toxicity.

Effect of Cr on the Mineral Structure and Composition of Cement Clinker and Its Solidification Behavior.

In order to reveal the solidification behavior of Cr in the cement clinker mineral phase, 29Si magic-angle spinning nuclear magnetic resonance, X-ray diffraction, and scanning electron microscopy with energy-dispersive X-ray spectroscopy techniques were used to analyze the morphology and composition of the cement clinker mineral phase doped with Cr. The results showed that the addition of Cr did not change the chemical environment of 29Si in the clinker mineral phase, and it was still an isolated silicon-oxygen tetrahedron. Cr affected the orientation of the silicon-oxygen tetrahedron and the coordination number of calcium, leading to the formation of defects in the crystal structure of the clinker mineral phase, by replacing Ca2+ into the mineral phase lattice to form a new mineral phase Ca3Cr2(SiO4)3. Cr acted as a stabilizer for the formation of ß-C2S in the clinker calcination. As the amount of Cr increased, the relative content of C3S decreased and the relative content of C2S increased. Further, Cr easily dissolved in C2S, while it was not found in C3S. This study is conducive to further research on the mechanism of heavy metal solidification in cement clinker. Furthermore, it is important to evaluate the environmental risk of heavy metals in the process of sludge disposal through cement kiln and promote the utilization of sludge resources and the sustainable development of the cement industry.

Characterizing Novel Modifications of a Therapeutic Protein Using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry, Sedimentation Velocity Analytical Ultracentrifugation, and Structural Modeling.

Heterogeneity of biopharmaceutical products is common due to various co- and post-translational modifications and degradation events that occur during the biological production process and throughout the shelf life. Product-related variants resulting from these modifications potentially affect a product's biological activity and safety, and thus, their detailed structure characterization is of great importance for successful development of protein therapeutics. Specifically, in this study, two novel low-level product variants in a recombinant therapeutic protein were characterized via chromatographic enrichment followed by proteolytic digestion and analysis using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). One of the variants was identified to be the therapeutic protein missing a 61-amino-acid fragment from its N-terminus. Consequently, the other variant was found to be the therapeutic protein carrying the 61-amino-acid long peptide. Furthermore, detailed structure at the modification site of the latter variant was determined as that amino group from the protein's N-terminus linked to side chain carbonyl carbon at Asp 61 residue of the peptide, based on the complementary information from collision induced dissociation and electron transfer dissociation MS/MS analysis. Results from sedimentation velocity analytical ultracentrifugation and computational structural modeling supported the hypothesis that formation of these two variants was a result of protein self-association. In dimeric state, the head-to-toe stacking conformation of two therapeutic protein molecules allowed spatial closeness between the N-terminus of one molecule and the 61st amino acid of the other molecule, resulting in a novel peptide transfer  between the two protein molecules.

Understanding the conformational impact of chemical modifications on monoclonal antibodies with diverse sequence variation using hydrogen/deuterium exchange mass spectrometry and structural modeling.

Chemical modifications can potentially induce conformational changes near the modification site and thereby impact the safety and efficacy of protein therapeutics. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a powerful analytical technique with high spatial resolution and sensitivity in detecting such local conformational changes. In this study, we utilized HDX-MS combined with structural modeling to examine the conformational impact on monoclonal antibodies (mAbs) caused by common chemical modifications including methionine (Met) oxidation, aspartic acid (Asp) isomerization, and asparagine (Asn) deamidation. Four mAbs with diverse sequences and glycosylation states were selected. The data suggested that the impact of Met oxidation was highly dependent on its location and glycosylation state. For mAbs with normal glycosylation in the Fc region, oxidation of the two conserved Met252 and Met428 (Kabat numbering) disrupted the interface interactions between the CH2 and CH3 domains, thus leading to a significant decrease in CH2 domain thermal stability as well as a slight increase in aggregation propensity. In contrast, Met oxidation in the variable region and CH3 domain had no detectable impact on mAb conformation. For aglycosylated mAb, Met oxidation could cause a more global conformational change to the whole CH2 domain, coincident with the larger decrease in thermal stability and significant increase in aggregation rate. Unlike Met oxidation, Asn deamidation and Asp isomerization mostly had very limited effects on mAb conformation, with the exception of succiminide intermediate formation which induced a measurable local conformational change to be more solvent protected. Structural modeling suggested that the succinimide intermediate was stabilized by adjacent aromatic amino acids through ring-ring stacking interactions.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

Interaction of cholesterol-conjugated ionizable amino lipids with biomembranes: lipid polymorphism, structure-activity relationship, and implications for siRNA delivery.

Delivery of siRNA is a major obstacle to the advancement of RNAi as a novel therapeutic modality. Lipid nanoparticles (LNP) consisting of ionizable amino lipids are being developed as an important delivery platform for siRNAs, and significant efforts are being made to understand the structure-activity relationship (SAR) of the lipids. This article uses a combination of small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) to evaluate the interaction between cholesterol-conjugated ionizable amino lipids and biomembranes, focusing on an important area of lipid SAR--the ability of lipids to destabilize membrane bilayer structures and facilitate endosomal escape. In this study, cholesterol-conjugated amino lipids were found to be effective in increasing the order of biomembranes and also highly effective in inducing phase changes in biological membranes in vitro (i.e., the lamellar to inverted hexagonal phase transition). The phase transition temperatures, determined using SAXS and DSC, serve as an indicator for ranking the potency of lipids to destabilize endosomal membranes. It was found that the bilayer disruption ability of amino lipids depends strongly on the amino lipid concentration in membranes. Amino lipids with systematic variations in headgroups, the extent of ionization, tail length, the degree of unsaturation, and tail asymmetry were evaluated for their bilayer disruption ability to establish SAR. Overall, it was found that the impact of these lipid structure changes on their bilayer disruption ability agrees well with the results from a conceptual molecular "shape" analysis. Implications of the findings from this study for siRNA delivery are discussed. The methods reported here can be used to support the SAR screening of cationic lipids for siRNA delivery, and the information revealed through the study of the interaction between cationic lipids and biomembranes will contribute significantly to the design of more efficient siRNA delivery vehicles.

Ionization behavior of amino lipids for siRNA delivery: determination of ionization constants, SAR, and the impact of lipid pKa on cationic lipid-biomembrane interactions.

Ionizable amino lipids are being pursued as an important class of materials for delivering small interfering RNA (siRNA) therapeutics, and research is being conducted to elucidate the structure-activity relationships (SAR) of these lipids. The pK(a) of cationic lipid headgroups is one of the critical physiochemical properties of interest due to the strong impact of lipid ionization on the assembly and performance of these lipids. This research focused on developing approaches that permit the rapid determination of the relevant pK(a) of the ionizable amino lipids. Two distinct approaches were investigated: (1) potentiometric titration of amino lipids dissolved in neutral surfactant micelles; and (2) pH-dependent partitioning of a fluorescent dye to cationic liposomes formulated from amino lipids. Using the approaches developed here, the pK(a) values of cationic lipids with distinct headgroups were measured and found to be significantly lower than calculated values. It was also found that lipid-lipid interaction has a strong impact on the pK(a) values of lipids. Lysis of model biomembranes by cationic lipids was used to evaluate the impact of lipid pK(a) on the interaction between cationic lipids and cell membranes. It was found that cationic lipid-biomembrane interaction depends strongly on lipid pK(a) and solution pH, and this interaction is much stronger when amino lipids are highly charged. The presence of an optimal pK(a) range of ionizable amino lipids for siRNA delivery was suggested based on these results. The pK(a) methods reported here can be used to support the SAR screen of cationic lipids for siRNA delivery, and the information revealed through studying the impact of pK(a) on the interaction between cationic lipids and cell membranes will contribute significantly to the design of more efficient siRNA delivery vehicles.

Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape.

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.

Formulation of a peptide nucleic acid based nucleic acid delivery construct.

Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within submicrometer-sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes. In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging 30-50 nm were created, with minimal dependence of complex size on N/P ratio (PEI amines to DNA phosphates). Furthermore, PEI-DNA interactions were altered by the derivatization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison to PEI-DNA complexes. Transfection experiments in Chinese hamster ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI-DP3 complexes relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery.

Towards development of stable formulations of a live attenuated bacterial vaccine: a preformulation study facilitated by a biophysical approach.

Development of optimal formulation conditions stabilizing live attenuated bacterial vaccines is impeded by traditional methods used for viability measurement. To facilitate preformulation studies of such vaccines, spectroscopic techniques capable of providing real-time and high throughput information have been employed to obtain a global stability profile for a live attenuated Ty21a bacterial typhoid vaccine over a wide range of pH (4 to 8) and temperature (10 to 85 degrees C). Using the data obtained from fluorescence and circular dichroism techniques, an empirical phase diagram (EPD) has been subsequently constructed, which suggests that Ty21a cells exist in at least four apparent physical phases related to different viability states, with the most stable phase at pH 6 and 7 at temperatures below 30 degrees C. A slightly basic pH (pH 8) appears to decrease the fluidity of the cell membrane, whereas acidic pH conditions are detrimental to membrane integrity over the entire temperature range. Based on the above stability profile, a fluorescence-based high throughput screening assay has been developed to test the stabilizing effects of various compounds at different concentrations. Amongst other promising stabilizers, 10% sucrose and 0.15 M glutamic acid display the greatest protective effects, with an increase of about 10 degrees C in the transition temperature of Ty21a cells. Preliminary studies have also been performed on foam dried formulations as an alternative approach to further stabilize Ty21a cells. The data show that 10% sucrose and trehalose both increase the in-process and storage stabilities of the cells.

Effects of pH and polyanions on the thermal stability of fibroblast growth factor 20.

Fibroblast growth factor 20 (FGF20) is a member of the FGF family with potential for use in several different therapeutic categories. In this work, we provide the first structural characterization of FGF20 using a wide variety of approaches. Like other members of the FGF family, FGF20 appears to possess a beta-trefoil structure. The effect of pH on the conformation and thermal stability of FGF20 is evaluated using far-UV circular dichroism (CD), intrinsic and ANS fluorescence, and high-resolution derivative UV absorption spectroscopy. Empirical phase diagrams are constructed to describe the solution behavior of FGF20 over a wide pH and temperature range. The protein appears to be unstable at pH <5, with aggregation and precipitation observed during dialysis. A major heat-induced conformational change also causes aggregation and precipitation of FGF20 at elevated temperatures. The highest thermal stability is observed near neutral pH (Tm ~55 degrees C at pH 7). The effect of several high- and low-molecular mass polyanions on the thermal stability of FGF20 is also examined using CD, intrinsic fluorescence, and DSC analysis. Among these ligands, heparin exhibits the greatest stabilizing effect on FGF20, increasing the Tm by more than 10 degrees C.

Effects of solutes on empirical phase diagrams of human fibroblast growth factor 1.

A variety of solutes are commonly used to increase the stability of protein in therapeutic formulations. An empirical phase diagram approach is used to evaluate the effects of different types of additives on the solution behavior of a protein of pharmaceutical interest, human fibroblast growth factor 1 (FGF-1). A specific stabilizer, heparin, and a nonspecific stabilizer, sucrose, were used in this work. The protein was characterized as a function of pH (3-8) and temperature (10-85 degrees C) using Far-UV circular dichroism (Far-UV CD), intrinsic and extrinsic fluorescence as well as second derivative UV absorption spectroscopy. Empirical phase diagrams were constructed to summarize the biophysical characterization data obtained with FGF-1 alone, in the presence of a threefold weight excess of heparin (3x heparin) or 10% sucrose (w/v). Three phases are observed in the low temperature regions at pH 3, 4, and 5-8. Phase boundaries corresponding to major heat-induced transitions are detected in the physiological temperature range. The highest thermal stabilities are observed near neutral pH (pH 6 and 7). Both heparin and sucrose appear to enhance the thermal stability of FGF-1, although their effects on the phase diagram are quite distinct. The greatest stabilization is observed at pH 8. Only heparin appears to protect FGF-1 from acid-induced unfolding to any extent.

Conformational lability of two molecular chaperones Hsc70 and gp96: effects of pH and temperature.

Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformational dynamics rather than immediate structural homology.

Solution behavior of IFN-beta-1a: an empirical phase diagram based approach.

An empirical phase diagram approach has been developed as a practical tool to aid macromolecular preformulation/formulation studies. This method employs an eigenvector based procedure to visualize and interpret complex data sets. Human Inteferon-beta-1a, an important therapeutic protein, was used to further develop the method and test its utility. The protein was characterized in solution as a function of pH (2-8), temperature (10 degrees C-85 degrees C) and ionic strength (I = 0.1 and 1.0) using intrinsic and ANS fluorescence, Far-UV circular dichroism (Far-UV CD), Fourier Transform Infrared spectroscopy (FTIR) and derivative UV absorbance spectroscopies, as well as differential scanning calorimetry (DSC) to supplement spectroscopic thermal stability studies. Derivative UV absorbance data were initially used to construct a pH-temperature phase diagram at each ionic strength. Three distinctive phases at I = 0.1 and two major phases at I = 1.0 were identified corresponding to different conformation/aggregation states of the protein. For the first time, heterogeneous data sets (i.e., data from different techniques) including Far-UV CD, fluorescence and UV absorbance results were used to generate empirical phase diagrams. Results from different data sets are compared; precautions in applying the method and its overall utility are discussed.