Frozen Aqueous Solutions Research Articles

Accelerated Degradation of Microplastics at the Liquid Interface of Ice Crystals in Frozen Aqueous Solutions

AbstractMicroplastics (MPs) are one of the emerging contaminants in environmental media, and they have raised great concern because they are resistant to degradation and persist in ecosystems. Although numerous advanced technologies have been developed, suitable technologies are still lacking for degradation of widespread MPs in the natural environment. We have discovered that MPs can be degraded exceptionally rapidly in frozen environments. Taking polystyrene (PS) as an example, its degradation rate in ice (−20 °C) is surprisingly competitive to most artificial technologies. PS particles are trapped and squeezed to achieve excited state (3PS*) in the narrow space of the liquid layer between ice crystals, which further react with the highly concentrated dioxygen to selectively produce singlet oxygen (1O2). The 1O2 boosts PS oxidation in the liquid layer thus further causing accelerated degradation at freezing temperature. This finding offers a highly efficient pathway for degradation of MPs and it sheds light on an unusual MPs disposal mechanisms in nature.

Accelerated Degradation of Microplastics at the Liquid Interface of Ice Crystals in Frozen Aqueous Solutions.

Microplastics (MPs) are one of the emerging contaminants in environmental media, and they have raised great concern because they are resistant to degradation and persist in ecosystems. Although numerous advanced technologies have been developed, suitable technologies are still lacking for degradation of widespread MPs in the natural environment. We have discovered that MPs can be degraded exceptionally rapidly in frozen environments. Taking polystyrene (PS) as an example, its degradation rate in ice (-20 °C) is surprisingly competitive to most artificial technologies. PS particles are trapped and squeezed to achieve excited state (3 PS*) in the narrow space of the liquid layer between ice crystals, which further react with the highly concentrated dioxygen to selectively produce singlet oxygen (1 O2 ). The 1 O2 boosts PS oxidation in the liquid layer thus further causing accelerated degradation at freezing temperature. This finding offers a highly efficient pathway for degradation of MPs and it sheds light on an unusual MPs disposal mechanisms in nature.

Role of arginine salts in preventing freezing-induced increase in subvisible particles in protein formulations

While arginine hydrochloride (ArgHCl) has emerged as a potential stabilizer of protein drugs in liquid formulations, the purpose of this manuscript was to evaluate its stabilization potential in frozen solutions. The phase behavior of frozen ArgHCl solutions was investigated by differential scanning calorimetry and low temperature powder X-ray diffractometry. The aggregation of β-galactosidase was evaluated following freeze–thaw cycling in ArgHCl solutions with and without mannitol. ArgHCl (5% w/v) was retained amorphous in frozen aqueous solutions and effectively inhibited protein aggregation even after 5 freeze–thaw cycles. Annealing frozen arginine solution (5% w/v) containing mannitol (10% w/v) induced mannitol crystallization which in turn facilitated crystallization of ArgHCl. The stabilizing effect of ArgHCl was completely lost in the presence of mannitol. Use of alternate arginine salts (aspartate, glutamate, and acetate) allowed selective crystallization of mannitol while arginine was retained amorphous and stabilized the protein.

ESEM Methodology for the Study of Ice Samples at Environmentally Relevant Subzero Temperatures: "Subzero ESEM".

Frozen aqueous solutions are an important subject of study in numerous scientific branches including the pharmaceutical and food industry, atmospheric chemistry, biology, and medicine. Here, we present an advanced environmental scanning electron microscope methodology for research of ice samples at environmentally relevant subzero temperatures, thus under conditions in which it is extremely challenging to maintain the thermodynamic equilibrium of the specimen. The methodology opens possibilities to observe intact ice samples at close to natural conditions. Based on the results of ANSYS software simulations of the surface temperature of a frozen sample, and knowledge of the partial pressure of water vapor in the gas mixture near the sample, we monitored static ice samples over several minutes. We also discuss possible artifacts that can arise from unwanted surface ice formation on, or ice sublimation from, the sample, as a consequence of shifting conditions away from thermodynamic equilibrium in the specimen chamber. To demonstrate the applicability of the methodology, we characterized how the true morphology of ice spheres containing salt changed upon aging and the morphology of ice spheres containing bovine serum albumin. After combining static observations with the dynamic process of ice sublimation from the sample, we can attain images with nanometer resolution.

Effect of Inorganic Phosphate on the Photoproduction of Hydrogen Peroxide in Frozen Aqueous Solutions of Adenine Derivatives

The content of hydrogen peroxide in 2 × 10−4 M aqueous solutions of adenine (A), adenosine (Ado) and adenosine-5'-diphosphate (ADP) containing NaCl (0.1 M) irradiated with near-UV light at 77 K and identical solutions containing added inorganic phosphate (Pi) is determined. It is shown that introducing Pi into the solutions considerably improves the yield of H2O2. The results from detecting H2O2 in the studied systems are compared to estimates of the integral intensities of EPR signals (IntS) of the irradiated solutions before defrosting (and the estimates of content of different components of these spectra, obtained by modeling). It is concluded that the basic producers of H2O2 in the studied systems are aggregates of adenine derivatives that form upon the freezing of aqueous solutions.

Control of Solvent Dynamics around the B12-Dependent Ethanolamine Ammonia-Lyase Enzyme in Frozen Aqueous Solution by Using Dimethyl Sulfoxide Modulation of Mesodomain Volume.

The temperature-dependent structure and dynamics of two concentric solvent phases, the protein-associated domain (PAD) and the mesodomain, that surround the ethanolamine ammonia-lyase (EAL) protein from Salmonella typhimurium in frozen polycrystalline aqueous solution are addressed by using electron paramagnetic resonance spectroscopy of the paramagnetic nitroxide spin probe, TEMPOL, over the temperature ( T) range 190-265 K. Dimethyl sulfoxide (DMSO), added at 0.5, 2.0, and 4.0% v/v and present at the maximum freeze concentration at T ≤ 245 K, varies the volume of the interstitial aqueous DMSO mesodomain ( Vmeso) relative to a fixed PAD volume ( VPAD). The increase in Vmeso/ VPAD from 0.8 to 6.0 is quantified by the partitioning of TEMPOL between the two phases. As Vmeso/ VPAD is increased, the Arrhenius parameters for activated TEMPOL rotational motion in the mesodomain remain uniform, whereas the parameters for TEMPOL in the PAD show a progressive transformation toward the mesodomain values (higher mobility). An order-disorder transition (ODT) in the PAD is detected by the exclusion of TEMPOL from the PAD into the mesodomain. The ODT T value is systematically lowered by increased Vmeso/ VPAD (from 215 to 200 K), and PAD ordering kinks the mesodomain Arrhenius dependence. Thus there is reciprocity in PAD-mesodomain solvent coupling. The results are interpreted as a dominant influence of ice-boundary confinement on the PAD solvent structure and dynamics, which is transmitted through the mesodomain and which decreases with mesodomain volume at increased added DMSO. The systematic tuning of PAD and mesodomain solvent dynamics by the variation of added DMSO is an incisive approach for the resolution of contributions of protein-solvent dynamical coupling to EAL catalysis.

Effect of Zn2+ and Ca2+ Ions on Hydrogen Peroxide Photoproduction in Frozen Aqueous Solutions of Adenine Derivatives

The content of hydrogen peroxide in aqueous 2 × 10–4 M solutions of adenine (A), adenosine (Ado), and adenosine-5'-diphosphate (ADP) containing NaCl (0.1 M) and identical solutions containing ZnCl2 and CaCl2 additions near UV-irradiated at 77 K was determined. The introduction of bivalent metal ions (2 × 10–4 M) differently affected the yield of H2O2 in different compounds. In A solutions, the addition of Zn2+ ions led to a yield of H2O2 increased more than 20-fold, and the addition of Ca2+ led to a 1.4-fold increase. In the case of Ado, the yield of H2O2 decreased 2.2- and 2.9-fold when Zn2+ and Ca2+ ions, respectively, were introduced in solutions. The addition of Zn2+ to ADP solutions led to a ~1.8-fold increase in the yield of H2O2. A comparison of the results of H2O2 determination in the systems with the estimated integral intensities of the EPR signals (IntS) of the irradiated solutions before defreezing (and estimated contents of the components of these spectra obtained by simulation) indicated that the main products of H2O2 formation in the systems were aggregates of adenine derivatives that formed during the freezing of aqueous solutions.

Histidine-epoxy-activated sepharose beads embedded poly (2-hydroxyethyl methacrylate) cryogels for pseudobiospecific adsorption of human immunoglobulin G

ABSTRACTThe use of highly purified immunoglobulin became among the most powerful adopted strategies in therapeutic trials nowadays. Their role as immunomodulatory and anti-inflammatory agents has widened their scope of use. A novel continuous supermacroporous monolithic cryogels embedded with histidine-epoxy-activated-sepharose beads were synthetized as a new monolithic adsorbents for the separation of immunoglobulin G from human serum. The histidine-epoxy-activated-sepharose beads were embedded into the 2-hydroxyethyl methacrylate (HEMA) cryogels present in frozen aqueous solution inside a plastic syringe. The microstructure morphology of the cryogels was characterized by swelling measurement and scanning electron microscopy. The adsorption of human IgG on the histidine-epoxy-activated-sepharose beads pHEMA cryogels appeared to follow the Langmuir–Freundlich adsorption isotherm model. The maximum IgG adsorption was observed at 4°C and pH 7.4 and was found to be 26.95 mg/g of cryogel which is close to that obtained experimentally (24.49 mg/g). The cryogels were used for several adsorption-desorption cycles without any negligible decrease in their adsorption capacity.

Amorphous-Amorphous Phase Separation of Freeze-Concentrated Protein and Amino Acid Excipients for Lyophilized Formulations.

The objective of this study was to elucidate the mixing state of proteins and amino acid excipients concentrated in the amorphous non-ice region of frozen solutions. Thermal analysis of frozen aqueous solutions was performed in heating scans before and after a heat treatment. Frozen aqueous solutions containing a protein (e.g., recombinant human albumin, gelatin) or a polysaccharide (dextran) and an amino acid excipient (e.g., L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) at varied mass ratios showed single or double Tg' (glass transition temperature of maximally freeze-concentrated solutes). Some mixture frozen solutions rich in the polymers maintained the single Tg' of the freeze-concentrated amorphous solute-mixture phase. In contrast, amino acid-rich mixture frozen solutions revealed two Tg's that suggested transition of concentrated non-crystalline solute-mixture phase and excipient-dominant phase. Post-freeze heat treatment induced splitting of the Tg' in some intermediate mass ratio mixture solutions. The mixing state of proteins and amino acids varied depending on their structure, salt types, mass ratio, composition of co-solutes (e.g., NaCl) and thermal history. Information on the varied mixing states should be valuable for the rational use of amino acid excipients in lyophilized protein pharmaceuticals.

Goldanskii–Karyagin effect on hyperalkaline tin(II)-hydroxide

Frozen aqueous solution of hyperalkaline tin(II)-hydroxide was analysed by 119Sn Mossbauer spectroscopy at low temperature in order to determinate the structure of the hydroxo complex formed under hyperalkaline (pH > 13) conditions. Interestingly, the quadrupole doublet characteristic of this complex in the 119Sn Mossbauer spectrum exhibited asymmetry in the line intensities. Analysis of the temperature dependence of the Mossbauer spectra demonstrated that this phenomenon can be rationalised by the Goldanskii–Karyagin effect. The effect emerges due to the vibrational anisotropy of bonds in the tin complex formed in hyperalkaline solution, similarly to what has been found earlier for SnF2 with analogous Sn bond structure.

Enhanced Removal of Hexavalent Chromium in the Presence of H2O2 in Frozen Aqueous Solutions.

The reductive transformation of Cr(VI) to Cr(III) by H2O2 in ice was compared with that in water. The reduction of Cr(VI) was significant at -20 °C (ice), whereas the reduction efficiency was very low at 25 °C (water). This enhanced reduction of Cr(VI) in ice was observed over a wide range of H2O2 concentration (20-1000 μM), pH (3-11), and freezing temperature (-10 to -30 °C). The observed molar ratio of consumed [H2O2] to reduced [Cr(VI)] in ice was in close agreement with the theoretical (stoichiometric) molar ratio (1.5) for H2O2-mediated Cr(VI) reduction through proton-coupled electron transfer (PCET). The synergistic increase in Cr(VI) reduction in water by increasing the H2O2 and proton concentrations confirms that the freeze concentration of both H2O2 and protons in the liquid brine is primarily responsible for the enhanced Cr(VI) reduction in ice. In comparison, the one-electron reduction of Cr(VI) to Cr(V) and subsequent reoxidation of Cr(V) to Cr(VI) is the major reaction mechanism in aqueous solution. The reduction efficiency of Cr(VI) by H2O2 in the frozen aqueous electroplating wastewater was similar to that in the frozen aqueous deionized water, which verifies the enhanced reduction of Cr(VI) by freezing in real Cr(VI)-contaminated aquatic systems.

The Photochemistry of Thymine in Frozen Aqueous Solution: Trimeric and Minor Dimeric Products

Early work identified three compounds, namely the c,s cyclobutane dimer, the so-called (6-4) photoproduct (5-hydroxy-6-4'-(5-methylpyrimidin-2'-one)-5,6-dihydrothymine) and a trimer hydrate, as products formed upon UV irradiation of thymine in frozen aqueous solution. More recent work has shown that an (α-4) product, namely α-4'-(5'-methylpyrimidine-2'-one)-thymine, is a likely product formed under these reaction conditions. During a thorough reinvestigation of the photochemistry of Thy in ice at -78.5°C, we found that a variety of other products could be detected. In addition to the c,s dimer, the other three known cyclobutane dimers, namely the c,a, t,s and t,a forms, are produced, although in considerably smaller amounts. The so-called "spore product" of thymine (5,6-dihydro-5-(α-thyminyl)thymine) is likewise formed. Two other dimers have been identified as minor products; one of these has been determined to be 5-(thymin-3-yl)-5,6-dihydrothymine and the other has been tentatively assigned to be a (5-4) adduct (6-hydroxy-5-4'-(5-methylpyrimidin-2'-one)-5,6-dihydrothymine). Compounds with the behavior expected of true trimeric compounds have been isolated via HPLC and characterized by mass spectrometry and photochemical behavior. One of these materials, putatively containing an oxetane ring, decomposes thermally to a secondary trimeric product that is then converted into the known trimer hydrate.

Physical Characterization of Pentamidine Isethionate during Freeze-Drying—Relevance to development of Stable Lyophilized Product

The purpose of this study was to perform physical characterization of pentamidine isethionate (PI) in frozen and freeze-dried systems and to monitor the phase behavior during all the stages of freeze-drying. Frozen aqueous PI solutions as well as the final lyophiles were characterized by differential scanning calorimetry and X-ray diffractometry. The effect of cosolutes, cosolvents, and processing conditions on the PI crystallization behavior during freeze-drying was evaluated. In frozen aqueous solutions, irrespective of the cooling rate and the initial solute concentration, PI readily crystallized as a trihydrate (C(19) H(24) N(4) O(2) ·3H(2) O). It dehydrated to a poorly crystalline anhydrate upon drying at 100 mTorr. The presence of a readily crystallizing cosolute or an organic cosolvent did not influence the physical form of PI in the final lyophile. On the contrary, even in the absence of cosolutes and cosolvents, the crystalline trihydrate was retained when the chamber pressure was increased to 500 mTorr. By altering the drying conditions, it was possible to obtain either a crystalline trihydrate or a poorly crystalline anhydrate. The stability of PI is dependent on its physical form and only the amorphous PI undergoes discoloration. The PI stability can be enhanced by retaining it in a crystalline state in the lyophile.

Impact of Heat Treatment on the Physical Properties of Noncrystalline Multisolute Systems Concentrated in Frozen Aqueous Solutions

The purpose of this study was to elucidate the effect of heat treatment on the miscibility of multiple concentrated solutes that mimic biopharmaceutical formulations in frozen solutions. The first heating thermal analysis of frozen solutions containing either a low-molecular-weight saccharide (e.g., sucrose, trehalose, and glucose) or a polymer (e.g., polyvinylpyrrolidone and dextran) and their mixtures from -70°C showed a single transition at glass transition temperature of maximally freeze-concentrated solution (T(g) ') that indicated mixing of the freeze-concentrated multiple solutes. The heat treatment of single-solute and various polymer-rich mixture frozen solutions at temperatures far above their T(g) ' induced additional ice crystallization that shifted the transitions upward in the following scan. Contrarily, the heat treatment of frozen disaccharide-rich solutions induced two-step heat flow changes (T(g) ' splitting) that suggested separation of the solutes into multiple concentrated noncrystalline phases, different in the solute compositions. The extent of the T(g) ' splitting depended on the heat treatment temperature and time. Two-step glass transition was observed in some sucrose and dextran mixture solids, lyophilized after the heat treatment. Increasing mobility of solute molecules during the heat treatment should allow spatial reordering of some concentrated solute mixtures into thermodynamically favorable multiple phases.

High-field EPR Study of the Effect of Chloride on Mn2+ Ions in Frozen Aqueous Solutions

The effect of chloride concentration on Mn2+ (S = 5/2, I = 5/2) ions in frozen aqueous solutions is studied by high-field high-frequency electron paramagnetic resonance (HFEPR). The usually six sharp lines characteristic of Mn2+ ions, arising from the m s = −1/2 → 1/2 transition, is modified by the addition of Cl− anions and the six resonances become much broader and more complex. This new feature likely arises from the ligation of one Cl− anion to a hydrated Mn2+ ion forming a [Mn(H2O)5Cl]− complex. This complex increases linearly with Cl− concentration with an association constant of K a, apparent = 61 M−1. The structure of the putative chloride complex was studied using density functional theory calculations and the expected zero-field interaction of such a manganese center was calculated using the superposition model. The predicted values were similar to those determined from the simulation of the spectrum of the m s = −5/3 → −3/2 transition of the chloride complex. This effect of Cl− anions occurs at biologically relevant concentration and can be used to probe the Mn2+ ions in cellular and protein environments.

Reaction of the CoII-Substrate Radical Pair Catalytic Intermediate in Coenzyme B12-Dependent Ethanolamine Ammonia-Lyase in Frozen Aqueous Solution from 190 to 217 K

The decay kinetics of the aminoethanol-generated CoII-substrate radical pair catalytic intermediate in ethanolamine ammonia-lyase from Salmonella typhimurium have been measured on timescales of <105 s in frozen aqueous solution from 190 to 217K. X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy of the disordered samples has been used to continuously monitor the full radical pair EPR spectrum during progress of the decay after temperature step reaction initiation. The decay to a diamagnetic state is complete and no paramagnetic intermediate states are detected. The decay exhibits three kinetic regimes in the measured temperature range, as follows. i), Low temperature range, 190≤T≤207K: the decay is biexponential with constant fast (0.57±0.04) and slow (0.43±0.04) phase amplitudes. ii), Transition temperature range, 207<T<214K: the amplitude of the slow phase decreases to zero with a compensatory rise in the fast phase amplitude, with increasing temperature. iii), High temperature range, T≥214K: the decay is monoexponential. The observed first-order rate constants for the monoexponential (kobs,m) and the fast phase of the biexponential decay (kobs,f) adhere to the same linear relation on an lnk versus T−1 (Arrhenius) plot. Thus, kobs,m and kobs,f correspond to the same apparent Arrhenius prefactor and activation energy (logAapp,f (s−1)=13.0, Ea,app,f=15.0 kcal/mol), and therefore, a common decay mechanism. We propose that kobs,m and kobs,f represent the native, forward reaction of the substrate through the radical rearrangement step. The slow phase rate constant (kobs,s) for 190≤T≤207K obeys a different linear Arrhenius relation (logAapp,s (s−1)=13.9, Ea,app,s=16.6 kcal/mol). In the transition temperature range, kobs,s displays a super-Arrhenius increase with increasing temperature. The change in Ea,app,s with temperature and the narrow range over which it occurs suggest an origin in a liquid/glass or dynamical transition. A discontinuity in the activation barrier for the chemical reaction is not expected in the transition temperature range. Therefore, the transition arises from a change in the properties of the protein. We propose that a protein dynamical contribution to the reaction, which is present above the transition temperature, is lost below the transition temperature, owing to an increase in the activation energy barrier for protein motions that are coupled to the reaction. For both the fast and slow phases of the low temperature decay, the dynamical transition in protein motions that are obligatorily coupled to the reaction of the CoII-substrate radical pair lies below 190K.

Photodecarbonylation of Dibenzyl Ketones and Trapping of Radical Intermediates by Copper(II) Chloride in Frozen Aqueous Solutions

This paper presents a quantitative and qualitative study of the Norrish type I reaction of dibenzyl ketone (DBK) and 4-methyldibenzyl ketone (MeDBK), producing the benzyl radicals and consequently recombination products, in frozen aqueous solutions over a broad temperature range (-80 to 20 degrees C). This work extends previous research on the cage effects in various constrained media to provide information about the dynamics and reactivity of the photochemically generated intermediates at the grain boundaries of ice matrix. As the temperature of aqueous solutions decreases, the solute concentrations become high at layers covering ice crystals, causing efficient molecular segregation. The cage effect experiments have shown that diffusion of the benzyl radicals within such reaction aggregates is still remarkably efficient at temperatures below -50 degrees C, independently of the initial ketone concentration in the range of 10(-6)-10(-4) mol L(-1). In addition, the study of trapping the benzyl radicals formed in situ by CuCl2 was used as a qualitative probe of heterogeneous bimolecular reactions in the frozen aqueous matrix and on its surface. Molecules of both solutes were found to be segregated from the ice phase to the same location and underwent chemical reactions within diffusion and intermediates lifetimes limits. Understanding the fundamental physicochemical processes in ice is unquestionably important in related environmental or cosmochemical investigations.

Weakly bound hydrogen and deuterium pairs in UV-photosensitized frozen aqueous solutions of KH2PO4 and KD2PO4

Weakly bound hydrogen and deuterium pairs in UV-photosensitized frozen aqueous solutions of KH2PO4 and KD2PO4

Solute Crystallization in Mannitol–Glycine Systems—Implications on Protein Stabilization in Freeze‐Dried Formulations

The use of mannitol in combination with glycine has resulted in stable freeze‐dried protein formulations. Our objectives were to (1) study solute crystallization in ternary systems containing mannitol, glycine, and water during all the stages of freeze drying as a function of processing conditions and formulation variables; (2) investigate the effect of sodium phosphate buffer salts on the crystallization of both mannitol and glycine and vice versa; and (3) investigate the effects of these excipients in a freeze‐dried lactate dehydrogenase (LDH) formulation. X‐ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study the frozen aqueous solutions. Phase transitions during primary and secondary drying were monitored by simulating the entire freeze‐drying process in situ in the sample chamber of the diffractometer. LDH activity after freeze drying was determined spectrophotometrically. In frozen aqueous solutions containing mannitol and glycine, each solute influenced the extent of crystallization of the other. The solutes crystallized as δ‐mannitol and β‐glycine during primary drying. Glycine had a stronger tendency to crystallize, while it was easier to influence mannitol crystallization. The buffer salts inhibited the crystallization of mannitol and glycine. However, in some cases, during primary drying, glycine crystallization was followed by that of disodium hydrogen phosphate dodecahydrate. The latter underwent dehydration forming an amorphous anhydrate. It was possible to correlate the extent of crystallization of mannitol and glycine in the lyophile with the retention of protein activity. An increase in buffer concentration decreased the crystallinity of mannitol and glycine. This translated to increased retention of protein activity. © 2003 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2272–2283, 2003

Enzymatic Peptide Synthesis in Frozen Aqueous Solution: Use of Nα-unprotected Peptide Esters as Acyl Donors

The ability of the endopeptidase α-chymotrypsin (EC 3.4.21.1) to catalyse the reaction of various Nα- unprotected di- and tripeptide ester derivatives with H-Leu-NH2, and with a series of C-terminal free di- and tripeptides at −15° C in frozen aqueous solution was investigated. The enzyme is able to synthesize N- and C-terminal unprotected penta- and hexapeptides in up to 92% yield, depending on the amino component used, in a single-step segment-condensation reaction. Freezing the reaction mixture resulted in significantly increased peptide yields compared with the reaction at room temperature. The enzyme shows a modified nucleophilic specificity in frozen solution compared with room temperature. Nucleophilic amino components with positively charged amino acids in P2′ -position are accepted. © 1997 European Peptide Society and John Wiley & Sons, Ltd.