The technology in cryotechnology.

The process of freezing biological material at extremely low temperatures is known as cryopreservation. To ensure the preservation of cells and tissues over an extended period of time, low temperatures are applied since biological processes, including the biochemical ones, come to a halt under cryogenic conditions and thus it is possible to maintain their structural and functional integrity. The field of cryopreservation gained more prominence in the 20th century and emerged as an unavoidable technology for different applications such as cell therapy, tissue engineering, or assisted fertilization. In this work we provide an overview of various technologies in the field of cryotechnology with regard to the freezing, storage and thawing of living cells. The first part covers the freezing process, starting with cryoprotective agents regarding their protection mechanisms and compositions, passing by cryo-imaging, micro-fluidic systems, and the currently available freezing and biobanking equipment. The second part focusses on the thawing process as well as the hypothermic preservation for the short-term storage of biological materials and constructs. Doi.org/10.54680/fr23610110112.

A spectroscopic and thermal investigation into the relationship between composition, secondary structure and physical characteristics of electrospun zein nanofibers.

Electrospun zein nanofibers have attracted interest as drug delivery systems due to their propensity for controlled drug release, flexible structure and low toxicity. However, comparatively little is known regarding the relationship between production method and fiber characteristics, both in terms of fiber architecture and protein structure. Here we use a range of imaging and spectroscopic techniques to elucidate the effects of solvent composition on zein secondary structure, fiber diameter and fiber integrity, plus we utilize the new technique of transition temperature microscopy to examine the thermal properties of the fibers. Zein nanofibers were prepared using ethanol, acetic acid and water mixes as solvents, alone and with plasticizers (polyethylene glycol, glycerol) and casein. Electrospinning was performed under controlled conditions and the products characterized using scanning electron microscopy (SEM), attenuated total reflection Fourier Transform infrared spectrometry (ATR - FTIR) and transition temperature microscopy (TTM). The choice of solvent, concentration and voltage, alongside the presence of additives (plasticizers and casein) were noted to influence both the diameter of the fibers and the tendency for bead formation. A relationship was noted between protein secondary structure and fiber architecture, with an enhanced ß-sheet content, enhanced by the inclusion of casein, being associated with higher beading. In addition, thermal imaging of electrospun zein fiber mats was successfully achieved using TTM via two dimensional mapping of the softening temperatures across the spun samples, in particular demonstrating the plasticizing effects of the polyethylene glycol and glycerol.

Identification and molecular interpretation of the effects of drug incorporation on the self-emulsification process using spectroscopic, micropolarimetric and microscopic measurements.

Addition of a drug to a self-emulsifying drug delivery system (SEDDS) can affect the emulsification process after administration, leading to variation in the emulsion droplet size formed and potentially its clinical behavior (Mercuri et al., Pharm. Res., 2011, 28, 1540-1551). However, the mechanisms involved and, in particular, the location of the drug within the system are poorly understood. Here, we have investigated the location of a model drug, ibuprofen, in the emulsions formed from a simple anhydrous SEDDS (soybean oil, Tween 80 and Span 80), using a range of physical characterization techniques. (1)H NMR studies showed an interaction between the drug and the polyoxyethylene chains of the surfactant Tween 80. Micropolarity assessment of the emulsion droplet interfacial region, using the chemical probes pyrene and Reichardt's dye, confirmed this interaction, and suggested that the drug was altering the microenvironment around the surfactants, and hence the behavior of the SEDDS with water during emulsification. Both dielectric spectroscopy and polarized light microscopy highlighted the differential behavior with water of placebo and drug-loaded SEDDS, also seen in the initial visual observational studies on the emulsification performance of the SEDDS. (1)H NMR studies with three other NSAIDs indicate that this effect is not confined to ibuprofen alone. The study has therefore indicated that the drug's influence on the emulsification process may be related to interactions within the microenvironment of the surfactant layer. Furthermore, such interactions may be usefully identified and characterized using a combination of micropolarity, spectroscopic and microscopic methods.

Pharmacokinetics of the injectable formulation of methadone hydrochloride administered orally in horses.

Methadone hydrochloride is a synthetic mu-opioid receptor agonist with potent analgesic properties. Oral methadone has been successfully used in human medicine and may overcome some limitations of other analgesics in equine species for producing analgesia with minimal adverse effects. However, there are no studies describing the pharmacokinetics (PK) of oral opioids in horses. The aim of this study was to describe the PK of orally administered methadone (0.1, 0.2 and 0.4 mg/kg) and physical effects in 12 healthy adult horses. Serum methadone concentrations were measured by gas chromatography/mass spectrometry at predetermined time points for 24 h, and PK parameters were estimated using a noncompartmental model. Physical effects were observed and recorded by experienced clinicians. No drug toxicity, behavioural or adverse effects were observed in the horses. The disposition of methadone followed first order elimination and a biphasic serum profile with rapid absorption and elimination phases. The PK profile of methadone was characterized by high clearance (Cl/F), small volume of distribution (V(d)/F) and short elimination half-life (t(1/2)). The mean of the estimated t(1/2) (SD) for each dose (0.1, 0.2 and 0.4 mg/kg) was 2.2 (35.6), 1.3 (46.1) and 1.5 (40.8), and the mean for the estimated C(max) (SD) was 33.9 (6.7), 127.9 (36.0) and 193.5 (65.8) respectively.

The formation of aminorex in racehorses following levamisole administration. A quantitative and chiral analysis following synthetic aminorex or levamisole administration vs. aminorex-positive samples from the field: a preliminary report.

Beginning in 2004, the horseracing industry experienced an epidemic of drug positives for the amphetamine-like drug aminorex. Investigation of the therapeutic treatment of the horses called positive for this drug suggested that its source was from the administration of the anthelmintic levamisole. This study examines the urine concentrations of aminorex as a function of time following administration of synthetic, racemic aminorex. Confirmation of the presence of aminorex in urine samples from the horses known to be treated with levamisole is also presented as are data concerning the concentrations of aminorex in positives called from the field and the corresponding concentrations of levamisole found in the same samples. Furthermore, this study illustrates that the chiral isomer distribution of aminorex found in samples from the field is significantly different from that arising from the administration of synthetic, racemic aminorex and is similar to that observed from aminorex arising from levamisole administration. An examination of the chiral isomer distribution of aminorex and a determination of the presence of levamisole in a sample may be used to assess the source of an aminorex positive, distinguishing it from an intentional synthetic, racemic aminorex administration. The role of levamisole in aminorex formation is also discussed.

Cefquinome concentrations in endometrium after intrauterine treatment of cobactan 4.5% in mares and inflammatory response of the endometrium to this treatment.

This study was conducted to measure the concentration of cefquinome in the endometrium of mares after intrauterine treatment and to evaluate associated inflammation. Mares (n = 14) were randomly assigned to one of the following groups: (i) control (n = 4) were either not treated (n = 2) or received (n = 2) lactated Ringer's intrauterine for 1 or 3 days; (ii) treated mares (n = 10) received intrauterine cefquinome for 1 or 3 days. After at least 10 days had passed following the last treatment and ovulation, mares were given Prostaglandin F2alpha (PGF2alpha) and were randomly assigned to an alternate treatment. Endometrial biopsy samples were taken at 2, 8, 24 and 48 h, or at 4, 12 and 36 h, after the last treatment. Biopsy samples were taken at the same time points from control mares (n = 2) and lactated Ringer-treated mares (n = 2). Cefquinome concentrations were quantified using a high-performance liquid chromatography (HPLC) assay and inflammation was assessed using haematoxylin and eosin (H&E)-stained sections. Concentrations of cefquinome [559 (1 day) and 595 microg/g (3 days) at 2 h, and 403 (1 day) and 370 microg/g (3 days) at 4 h] were similar between treatment groups at 2 and 4 h after treatment (p > 0.05). At 8 h, as well as at 24 and 48 h, concentrations were greater in the 3-day group (17 vs 301 microg/g, 3 vs 80 microg/g and 0.1 vs 0.2 microg/g, respectively) (p < 0.05). No significant differences (p > 0.05) in the inflammatory response at 2-48 h after treatment were found between groups.

Drug contamination of the equine racetrack environment: a preliminary examination.

Advances in analytical technology now make it feasible to detect and confirm exceptionally low concentrations (pg to fg/mL) of drugs and their metabolites in equine biological fluids. These new capabilities complicate the regulatory interpretation of drug positives and bring into question the fair application of medication rules. Such approaches and policies are further complicated by the possibility that drug positives may arise from contamination of the equine environment on the backstretch of the race track. This manuscript provides data demonstrating that the general environment of the backstretch in which horses live is contaminated with therapeutic drugs and drugs of human origin. The major contaminants are nonsteroidal anti-inflammatory drugs, such as flunixin, phenylbutazone and naproxen, present in the soil in stalls, on stall surfaces, in the dust that circulates and in the lagoon waters that accumulate on the backstretch. The presence of caffeine and cotinine suggest other possible vectors for contamination by humans. Concentrations of these compounds as well as their frequency of occurrence are provided.

Estrogen treatment increases the levels of regulator of G protein signaling-Z1 in the hypothalamic paraventricular nucleus: possible role in desensitization of 5-hydroxytryptamine1A receptors.

Desensitization of post-synaptic serotonin1A (5-HT1A) receptors may underlie the clinical improvement of neuropsychiatric disorders. In the hypothalamic paraventricular nucleus, Galphaz proteins mediate the 5-HT1A receptor-stimulated increases in hormone release. Regulator of G protein signaling-Z1 (RGSZ1) is a GTPase-activating protein selective for Galphaz proteins. RGSZ1 regulates the duration of interaction between Galphaz proteins and effector systems. The present investigation determined the levels of RGSZ1 in the hypothalamic paraventricular nucleus of rats subjected to four different treatment protocols that produce desensitization of 5-HT1A receptors. These protocols include: daily administration of beta estradiol 3-benzoate (estradiol) for 2 days; daily administration of fluoxetine for 3 and 14 days; daily administration of cocaine for 7 or 14 days; and acute administration of (+/-)-1-(2,5 dimethoxy-4-iodophenyl)-2-amino-propane HCl (DOI; a 5-HT2A/2C receptor agonist). Estradiol treatment was the only protocol that increased the levels of RGSZ1 protein in the hypothalamic paraventricular nucleus in a dose-dependent manner (46%-132% over control). Interestingly, previous experiments indicate that only estradiol produces a decreased Emax of 5-HT1A receptor-stimulation of hormone release, whereas fluoxetine, cocaine and DOI produce a shift to the right (increased ED50). Thus, the desensitization of 5-HT1A receptors by estradiol might be attributable to increased levels of RGSZ1 protein. These findings may provide insight into the adaptation of 5-HT1A receptor signaling during pharmacotherapies of mood disorders in women and the well-established gender differences in the vulnerability to depression.

An investigation into the structure and bioavailability of alpha-tocopherol dispersions in Gelucire 44/14.

In this investigation we describe the preparation, physical characterisation and in vivo behaviour of solid dispersions of a liquid nutraceutical, alpha-tocopherol, in Gelucire 44/14 with a view to establishing whether dispersion in this matrix may provide a means of formulating a liquid drug in a solid dosage form while also improving the oral bioavailability. Using Vitamin E Preparation USP as the source of alpha-tocopherol, dispersions were prepared using a melt-fusion method with active loadings up to 50% (w/w) and characterised using differential scanning calorimetry and optical microscopy. Capsules containing 300 IU alpha-tocopherol were manufactured and the absorption profiles compared to a commercial soft gelatin capsule preparation in healthy human volunteers. Confocal laser scanning microscopy (CLSM) studies were performed in order to elucidate the mechanism by which drug release may be occurring. Differential scanning calorimetry studies indicated that the presence of the active had a negligible effect on the melting profile of the carrier, indicating limited miscibility between the two components, a conclusion supported by the microscopy studies. Similarly, the dispersions were shown to exhibit a glass transition corresponding to the incorporated drug, indicating molecular cooperativity and hence phase separation from the lipid base. Despite the phase separation, it was noted that capsules stored for 18 months under ambient conditions showed no evidence of leakage. Bioavailability studies in six healthy male volunteers indicated that the Gelucire 44/14 formulation showed an approximately two-fold increase in total alpha-tocopherol absorption compared to the commercial preparation. Confocal laser scanning microscopy studies indicated that, on contact with water, the dispersions formed two interfacial layers, from which the Gelucire 44/14 disperses in the liquid medium as small particles. Furthermore, evidence was obtained for the dispersed material becoming incorporated into the hydrated lipid. In conclusion, the dispersion of the liquid drug in Gelucire 44/14 appears to allow the dual advantages of the preparation of a solid formulation and improved bioavailability of this material.

RGSZ1 and Ret RGS: two of several splice variants from the gene RGS20.

RGSZ1 and Ret RGS, members of the regulator of G-protein signaling (RGS) family, are GTPase-activating proteins (GAPs) with high selectivity for G alpha(z). We show here that RGSZ1 and Ret RGSZ1 are products of two of several splice variants of one gene, RGS20. RGS20 spans approximately 107 kb and contains at least seven exons. Five exons account for RGSZ1, including a single exon distinct to RGSZ1 that encodes a newly identified amino-terminal region. The previously described open reading frame (ORF) and 3' untranslated region are encoded by four downstream exons that also encode about half of Ret RGS. The 5' end of the RGSZ1 ORF contains several in-frame ATG codons (3-5 depending on the species), and multiple translational start sites may help explain the molecular weight heterogeneity of purified bovine brain RGSZ. Ret RGS replaces the 24 N-terminal amino acid residues of RGSZ1 with a large, N-terminal region that initially distinguished the bovine Ret RGS from human and mouse RGSZ1. This N-terminal domain is encoded by two distinct 5' exons that are variably combined with the four downstream exons shared with RGSZ1 to produce at least six mRNAs. They encode proteins with N termini that vary in size, hydrophobicity, and the presence of a cysteine string. At least two mRNAs that include the exon that encodes the N-terminal region unique to RGSZ1 were found in brain and a few other tissues, but not retina. RGS20 thus can account for multiple G(z)-selective GAPs in different tissues.

Combustion products of 1,3-butadiene are cytotoxic and genotoxic to human bronchial epithelial cells.

Adverse health effects of airborne toxicants, especially small respirable particles and their associated adsorbed chemicals, are of growing concern to health professionals, governmental agencies, and the general public. Areas rich in petrochemical processing facilities (e.g., eastern Texas and southern California) chronically have poor air quality. Atmospheric releases of products of incomplete combustion (e.g., soot) from these facilities are not subject to rigorous regulatory enforcement. Although soot can include respirable particles and carcinogens, the toxicologic and epidemiologic consequences of exposure to environmentally relevant complex soots have not been well investigated. Here we continue our physico-chemical analysis of butadiene soot and report effects of exposure to this soot on putative targets, normal human bronchial epithelial (NHBE) cells. We examined organic extracts of butadiene soot by gas chromatography-mass spectrometry (GC-MS), probe distillation MS, and liquid chromatography (LC)-MS-MS. Hundreds of aromatic hydrocarbons and polycyclic aromatic hydrocarbons with molecular mass as high as 1,000 atomic mass units were detected, including known and suspected human carcinogens (e.g., benzo(a)pyrene). Butadiene soot particles also had strong, solid-state free-radical character in electron spin resonance analysis. Spin-trapping studies indicated that fresh butadiene soot in a buffered aqueous solution containing dimethylsulfoxide (DMSO) oxidized the DMSO, leading to CH(3)* radical formation. Butadiene soot DMSO extract (BSDE)-exposed NHBE cells displayed extranuclear fluorescence within 4 hr of exposure. BSDE was cytotoxic to > 20% of the cells at 72 hr. Morphologic alterations, including cell swelling and membrane blebbing, were apparent within 24 hr of exposure. These alterations are characteristic of oncosis, an ischemia-induced form of cell death. BSDE treatment also produced significant genotoxicity, as indicated by binucleated cell formation. The combination of moderate cytotoxicity and genotoxicity, as occurred here, can be pro-carcinogenic.

Suppression of arylamine toxicity in the Fischer-344 rat following ingestion of a complex mixture.

The toxic effects of a mixture of 2-aminoanthracene (2-AA), benzanthracene (BA), and dinitropyrene isomers (DNP), and the toxic effects of these compounds individually, were investigated in the Fischer-344 rat following dietary exposure via a powdered basal diet. Animals were sacrificed at 14-, 30-, and 80-days of dietary exposure. Exposure to dietary 2-AA alone induced anorexia, cachexia, variable mortality, and altered serum chemistry profiles in the F-344 rat. Reduced lymphocyte counts were also shown in rats exposed to 2-AA. A temporal pattern of effect of 2-AA dietary exposure was observed in the progression of hepatic lesions in exposed animals. Dietary exposure to either DNP isomers or BA at a 10-fold higher concentration in the diet, relative to 2-AA, did not induce detectable toxic responses. However, exposure of rats to a mixture of 2-AA, BA, and DNP isomers (100 mg/kg, 1.0 g/kg, and 1.0 g/kg of diet, respectively) resulted in the attenuation of toxic effects when compared to exposure of F-344 rats to 2-AA alone. These results indicate that the toxic effects of 2-AA are suppressed by co-administration of DNP and BA and suggest that compound interactions need to be considered when predicting the toxic potential of specific environmental pollutants.

Hepatic toxicity and recovery of Fischer 344 rats following exposure to 2-aminoanthracene by intraperitoneal injection.

Humans may be exposed to 2-aminoanthracene (2-AA), a substituted polycyclic aromatic hydrocarbon, and a recognized mutagen and carcinogen, through oral and respiratory routes from contact with a variety of environmental sources. For the present study, we sought to evaluate hepatic damage and recovery in Fischer 344 rats following multiple i.p. injections of 5 mg of 2-AA. Rats were injected weekly for up to 5 weeks. Subgroups were then allowed to recover for 1, 5, or 9 weeks, and biochemical and pathologic changes were evaluated. We observed that weight gains were reduced relative to controls for all groups receiving > or = 2 injections. Serum enzyme levels indicative of liver damage were evident and included alterations in serum aspartate aminotransferase, alkaline phosphatase, total protein, albumin, and globulin. These alterations usually returned to normal by 5 weeks following cessation of 2-AA administration. In contrast, histologic liver changes, including hepatocyte hypertrophy, biliary hyperplasia with oval cell proliferation, altered foci, nodular hyperplasia, and one hepatocellular adenoma became more severe with time. This experiment demonstrates patterns of hepatic damage and recovery in rats exposed to 2-AA.

Quantitation of adenine nucleotides in equine colonic mucosal tissue using high performance liquid chromatography.

The objectives were to use high performance liquid chromatography (HPLC) to validate an established method for adenine nucleotide separation in equine colonic mucosal tissue, to determine the inherent variability in the tissue and extraction method, and to determine the stability of ATP, ADP, and AMP in the tissue with time. Equine colonic mucosal tissue obtained from a single horse was immediately submersed in liquid nitrogen, and stored at -70 degrees C. Samples were lyophilized, extracted, and separated by HPLC. The limit of quantitation was 0.05 microg/mL. The coefficient of variation for the instrument was less than 10% for all nucleotides measured. When the tissue was not homogenized prior to sampling, there were significant differences in adenine nucleotide content between samples. However, when the tissue was homogenized prior to analysis, these differences were no longer significant. There was no significant decrease in ATP, ADP, or AMP content over a 54-day analysis period.

Distribution of the hallucinogens N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine in rat brain following intraperitoneal injection: application of a new solid-phase extraction LC-APcI-MS-MS-isotope dilution method.

A method for the solid-phase extraction (SPE) and liquid chromatographic-atmospheric pressure chemical ionization-mass spectrometric-mass spectrometric-isotope dilution (LC-APcI-MS-MS-ID) analysis of the indole hallucinogens N,N-dimethyltryptamine (DMT) and 5-methoxy DMT (or O-methyl bufotenin, OMB) from rat brain tissue is reported. Rats were administered DMT or OMB by the intraperitoneal route at a dose of 5 mg/kg and sacrificed 15 min post treatment. Brains were dissected into discrete areas and analyzed by the methods described as a demonstration of the procedure's applicability. The synthesis and use of two new deuterated internal standards for these purposes are also reported.

Low frequency dielectric investigations into the relaxation behavior of frozen polyvinylpyrrolidone-water systems.

The low frequency dielectric response of aqueous solutions containing 0, 1, 5, and 10% w/v polyvinylpyrrolidone (PVP) was studied to characterize the low temperature relaxation behavior of these systems. Complementary modulated temperature differential scanning calorimetry (MTDSC) studies allowed measurement of the glass transition temperature for these materials, corresponding to the behavior of the nonfrozen phase. Dielectric investigations in the frequency range of 10(6) to 10(-2) Hz were performed on the systems in the liquid state, with a Maxwell-Wagner response noted for both the PVP solutions and water. The solid-phase responses were studied over a range of temperatures down to -70 degrees C, with a relaxation peak observed for the PVP systems in the kilohertz region. The spectra were modeled using the Havriliak-Negami equation and the corresponding relaxation times were calculated, with a satisfactory fit to the Arrhenius equation noted. The calculated activation energies were similar to literature values for the dielectric relaxation of water. It is suggested that the dielectric response is primarily a reflection of the relaxation behavior of the water molecules in the nonfrozen fraction, thereby indicating that the dielectric technique may yield insights into specific components of frozen aqueous systems.

An investigation into the low temperature thermal behaviour of vitamin E preparation USP using differential scanning calorimetry and low frequency dielectric analysis.

The thermal and dielectric responses of Vitamin E Preparation USP have been examined to further understand the melting and solidification of this material. A TA Instruments 2920 Differential Scanning Calorimeter was used to examine the thermal response of the sample at a range of scanning speeds. Isothermal dielectric studies were performed using a Novocontrol Dielectric Spectrometer over a range of temperatures down to -70 degrees C and a frequency range of 10(6)-10(-2) Hz. The differential scanning calorimetry (DSC) studies showed an anomalous response whereby at slow heating rates (2 degrees C min(-1)) a small exotherm followed immediately by an endotherm was observed. This response was considerably diminished in magnitude at higher rates (5 degrees C min(-1)) and was not observed at the fastest heating rate of 10 degrees C min(-1). No thermal events were seen on cooling the sample to -60 degrees C. It was suggested that the material formed a glass on cooling, with a predicted transition temperature of approximately -100 degrees C. Further studies using a liquid nitrogen cooling system indicated that the system did indeed exhibit a glass transition, albeit at a higher temperature than predicted (ca -63 degrees C). Low frequency dielectric analysis showed a clear relaxation peak in the loss component, from which the relaxation time could be calculated using the Havriliak-Negami model. The relationship between the relaxation time and the temperature was studied and was found to follow the Vogel-Tammann-Fulcher (VTF) modification of the Arrhenius equation. It is therefore concluded that Vitamin E Preparation USP is a glass-forming material that exhibits kinetically-hindered recrystallisation and melting behaviour. The study has also indicated that DSC and low frequency dielectric analysis may be powerful complementary tools in the study of the low temperature behaviour of pharmaceuticals.

Matrix solid-phase dispersion.

Matrix solid-phase dispersion (MSPD) is a patented process, first reported in 1989, for conducting simultaneous disruption and extraction of solid and semi-solid samples. MSPD permits complete fractionation of the sample matrix components as well as the ability to selectively elute a single compound or several classes of compounds from the same sample. The method has been applied to the isolation of drugs in food animal tissues but has also found wide application in the analysis of herbicides, pesticides and pollutants from animal tissues, fruits, vegetables and other matrices. The present article provides a review of MSPD applications in these and related fields and discusses the factors known to affect MSPD methods. Both the practical and theoretical aspects of MSPD are also presented.