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S D Allison

Publications and source records attributed to S D Allison.

18 recordsLinked to original sources
PubMed2001

Analysis of self-assembled cationic lipid-DNA gene carrier complexes using flow field-flow fractionation and light scattering.

Self-assembled cationic lipid-DNA complexes have shown an ability to facilitate the delivery of heterologous DNA across outer cell membranes and nuclear membranes (transfection) for gene therapy applications. While the size of the complex and the surface charge (which is a function of the lipid-to-DNA mass ratio) are important factors that determine transfection efficiency, lipid-DNA complex preparations are heterogeneous with respect to particle size and net charge. This heterogeneity contributes to the low transfection efficiency and instability of cationic lipid-DNA vectors. Efforts to define structure-activity relations and stable vector populations have been hampered by the lack of analytical techniques that can separate this type of particle and analyze both the physical characteristics and biological activity of the resulting fractions. In this study, we investigated the feasibility of flow field-flow fractionation (flow FFF) to separate cationic lipid-DNA complexes prepared at various lipid-DNA ratios. The compatibility of the lipid-DNA particles with several combinations of FFF carrier liquids and channel membranes was assessed. In addition, changes in elution profiles (or size distributions) were monitored as a function of time using on-line ultraviolet, multiangle light scattering, and refractive index detectors. Multiangle light scattering detected the formation of particle aggregates during storage, which were not observed with the other detectors. In comparison to population-averaged techniques, such as photon correlation spectroscopy, flow FFF allows a detailed examination of subtle changes in the physical properties of nonviral vectors and provides a basis for the definition of structure-activity relations for this novel class of pharmaceutical agents.

DNA↗
PubMed2001

Maintenance of nonviral vector particle size during the freezing step of the lyophilization process is insufficient for preservation of activity: insight from other structural indicators.

The instability of nonviral vectors as liquid formulations has stimulated considerable interest in developing dehydrated formulations that would be resistant to shipping stresses and could be stored at room temperature. Recently, we reported that high sucrose/DNA ratios are capable of maintaining particle size during the freezing step of the lyophilization process and we suggested that the separation of individual particles within sugar matrices is responsible for the reported protection of nonviral vectors during the freezing step of a typical lyophilization protocol. The purpose of this study was to extend these observations to other nonviral vectors that incorporate different cationic components. Cationic lipid-based complexes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), with helper lipid cholesterol (Chol) or dioleoylphosphatidyl-ethanolamine (DOPE), showed similar protection by sucrose. Formulations of a polyethylenimine (PEI)-based vector required much higher excipient/DNA ratios for size protection compared with protamine- and lipid-based vectors. At low sucrose/DNA ratios, zeta potentials for all complexes were significantly lowered during freezing. Similar results were obtained at high sucrose/DNA ratios, except for DOTAP-DOPE-containing vectors which maintained zeta potential values comparable to unfrozen controls. The changes in zeta potential values indicate that complexes are altered during freezing despite the maintenance of particle size as determined by light scattering. Furthermore, these changes might explain the observed reduction in transfection activity and provide new information about the effects of physicochemical changes of nonviral vectors during the freezing step of lyophilization.

Animals↗
PubMed2000

Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: the particle isolation hypothesis.

The instability of nonviral vectors in aqueous suspensions has stimulated an interest in developing lyophilized formulations for use in gene therapy. Previous work has demonstrated a strong correlation between the maintenance of particle size and retention of transfection rates. Our earlier work has shown that aggregation of nonviral vectors typically occurs during the freezing step of the lyophilization process, and that high concentrations of sugars are capable of maintaining particle size. This study extends these observations, and demonstrates that glass formation is not the mechanism by which sugars protect lipid/DNA complexes during freezing. We also show that polymers (e.g., hydroxyethyl starch) are not capable of preventing aggregation despite their ability to form glasses at relatively high subzero temperatures. Instead, our data suggest that it is the separation of individual particles within the unfrozen fraction that prevents aggregation during freezing, i.e., the particle isolation hypothesis. Furthermore, we suggest that the relatively low surface tension of mono- and disaccharides, as compared to starch, allows phase-separated particles to remain dispersed within the unfrozen excipient solution, which preserves particle size and transfection rates during freezing.

Calorimetry, Differential Scanning↗
PubMed2000

Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran.

The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting T(g) of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and T(g) by freeze-drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in T(g) observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability.

Actins↗
PubMed2000

Mechanisms of protection of cationic lipid-DNA complexes during lyophilization.

Gene therapy using nonviral vectors offers advantages over viral methods. However, the instability of aqueous suspensions of cationic lipid-DNA complexes is a major problem that must be overcome to develop this therapeutic modality on a pharmaceutical scale. Disaccharides have been reported to protect lipid-DNA complexes during lyophilization, and recovery of transfection correlates with the retention of particle size. However, the mechanism by which disaccharides achieve this protection is not known. The purpose of this study was to investigate the protective mechanism by lyophilizing cationic lipid-DNA complexes with a variety of solutes that have different physical behaviors during the lyophilization process. In agreement with previous studies, disaccharides conferred protection to lipid-DNA complexes. By contrast, a large polymeric sugar, hydroxyethyl starch, did not protect as well. The level of protection by additives, such as mannitol, that crystallized during lyophilization was also less than that of the disaccharides, but some protection was nonetheless observed. These data suggest that water replacement plays a significant role in protecting complexes during lyophilization. A second mechanism that prevents aggregation by diluting complexes within freeze-concentrated solutions or dried cakes may also contribute to protection. Sample vitrification did not correlate with maintenance of transfection efficiency. Elucidation of the mechanism(s) by which cationic lipid-DNA complexes are protected during lyophilization will permit a rational approach to the development of stable, lyophilized formulations.

Animals↗
PubMed1999

Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding.

The nature of the interaction responsible for the inhibition of protein unfolding and subsequent damage by sugars during dehydration is unclear. The relationship between sample moisture content measured by coulometric Karl Fischer titration and the apparent moisture content predicted by the area of the protein side chain carboxylate band at approximately 1580 cm-1 in infrared spectra of dried protein-sugar samples was examined. For samples in which a high level of native protein structure was retained in the dried solid, the apparent moisture content predicted by the carboxylate band area was greater than the actual moisture content, indicating that protection results from direct sugar-protein hydrogen bonding and not entrapment of water at the protein surface. Further, we show that the degree of structural protection conferred by sucrose and trehalose apparent in second derivative, amide I infrared spectra, correlates with the extent of hydrogen bonding between sugar and protein. The failure of dextran to inhibit dehydration-induced lysozyme unfolding is shown to result from the inability of the polymer to hydrogen bond adequately to the protein. Therefore, formation of an amorphous phase alone is not sufficient to maintain protein structure during dehydration. Glucose hydrogen bonds to a high degree with dried lysozyme, but is incapable of inhibiting lyophilization-induced protein unfolding in the absence of an effective cryoprotectant. However, the addition of polyethylene glycol, which is known to protect proteins during freezing, but not drying, to glucose protected lysozyme structure during lyophilization. Together, these results show that hydrogen bonding between carbohydrate and protein is necessary to prevent dehydration-induced protein damage. However, hydrogen bonding alone is not sufficient to protect proteins during lyophilization in the absence of adequate freezing protection.

Animals↗
PubMed1998

Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition.

Limited stability impedes the development of industrial and pharmaceutical proteins. Dried formulations are theoretically more stable, but the drying process itself causes structural damage leading to loss of activity after rehydration. Lyophilization is the most common method used to dry proteins, but involves freezing and dehydration, which are both damaging to protein. We compared an air-drying method to freeze-drying to test the hypothesis that terminal dehydration is the critical stress leading to loss of activity. The secondary structure of air-dried and freeze-dried actin was analyzed by infrared spectroscopy and related to the level of activity recovered from the rehydrated samples. Actin dried by either method in the absence of stabilizers was highly unfolded and the capacity to polymerize was lost upon rehydration. The degree of unfolding was reduced by air-drying or freeze-drying actin with sucrose, and the level of activity recovered upon rehydration increased. The addition of dextran to sucrose improved the recovery of activity from freeze-dried, but not air-dried samples. Dextran alone failed to protect the structure and function of actin dried by either method, indicating that proteins are not protected from dehydration-induced damage by formation of a glassy matrix. In some cases, recovered activity did not correlate directly with the level of structural protection conferred by a particular additive. This result suggests that secondary structural protection during drying is a necessary but not sufficient condition for the recovery of activity from a dried protein after rehydration.

Actins↗
PubMed1996

Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B.

Structural differences between two genetic variants of bovine beta-lactoglobulins (type A and B) in aqueous solutions were characterized using Fourier transform infrared and circular dichroism spectroscopies. To probe differences in structural dynamics, the effects hydrogen-deuterium exchange were also compared for the two proteins. The infrared spectra recorded in H2O solution for the two proteins were nearly identical in the conformationlly sensitive amide I region. The only exceptions were small differences at the band ascribed to a high-wavenumber beta-sheet component near 1693 cm-1 and the band assigned to turns at 1684 cm-1. In contrast, when the proteins were prepared in D2O solution, marked spectral differences were observed at all regions ascribed to beta-sheet and turn structures. These differences are consistent with the structural differences of the two variants at amino acid residues 64 and 118, which are located at a turn and a beta-sheet structure, respectively, as revealed by X-ray crystallographic studies [Monaco et al. (1987) J. Mol. Biol. 197, 695-706]. The circular dichroism spectra for the two proteins were essentially identical, both before and after hydrogen-deuterium exchange. Therefore, hydrogen-deuterium exchange did not alter the proteins' secondary structure. The enhancement of the amide I spectral difference upon hydrogen-deuterium exchange was ascribed to the differences in the structural mobility of the two proteins. Since the rate of exchange was greater for variant A, it was concluded that this variant has greater structural mobility than variant B. These findings indicate that the combination of infrared spectroscopy and hydrogen-deuterium exchange has great potential in characterization of even subtle structural differences in proteins induced by naturally occurring point mutations and/or site-directed mutagenesis.

Animals↗
PubMed1996

Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration.

Studies of numerous proteins with infrared spectroscopy have documented that unfolding is a general response of unprotected proteins to freeze-drying. Some proteins that are unfolded in the dried solid aggregate during rehydration, whereas others refold. It has been proposed for the latter case that aggregation is avoided because refolding kinetically outcompetes intermolecular interactions. In contrast, with proteins that normally aggregate after rehydration, minimizing unfolding during freeze-drying with stabilizer has been shown to be needed to favor the recovery of native protein molecules after rehydration. The purpose of the current study was to examine first the opposite situation, in which a denaturant is used to foster additional unfolding in the protein population during freeze-drying. If the protein is not intrinsically resistant to aggregation under the study conditions (e.g., because of intermolecular charge repulsion) and the denaturant does not disrupt intermolecular interactions during rehydration, this treatment should favor aggregation upon rehydration. With infrared spectroscopy we found that at concentrations of the denaturant Na thiocyanate (NaSCN) that only slightly perturbed chymotrypsinogen secondary structure in solution before freeze-drying, there was a large increase in protein unfolding in the dried solid and in protein aggregation measured after rehydration. Bands assigned to intermolecular beta sheet were present in the spectra of samples dried with NaSCN, indicating that aggregation could also arise in the dried solid. By examining the protein structure in the frozen state, we determined that in the absence of NaSCN the protein remains native. NaSCN caused structural perturbations during freezing, without the formation of intermolecular beta sheet, that were intermediate to structural changes noted after freeze-drying. In contrast, samples treated in the presence of NaSCN and sucrose had native-like spectra in the frozen and dried states, and much reduced aggregation after rehydration. These results indicate that during freezing and drying the sugar can counteract and mostly reverse the structural perturbations induced by NaSCN before and during these treatments.

Animals↗
PubMed1996

Quantitation of the area of overlap between second-derivative amide I infrared spectra to determine the structural similarity of a protein in different states.

Maintaining a native-like structure of protein pharmaceuticals during lyophilization is an important aspect of formulation. Infrared spectroscopy can be used to evaluate the effectiveness of formulations in protecting the secondary structural integrity of proteins in the dried solid. This necessitates making quantitative comparisons of the overall similarity of infrared spectra in the conformationally sensitive amide I region. We initially used the correlation coefficient r, as defined by Prestrelski et al. (Biophys. J. 1993, 65, 661-671), for this quantitation. Occasionally, we noticed that the r value did not agree with a visual assessment of the spectral similarity. In some cases this was due to an offset in baselines, which led artifactually to an unreasonably low r value. Conversely, if the spectra were baseline corrected and there existed a large similarity between peak positions, but differences in relative peak heights, the r value would be unreasonably high. Our approach to avoiding these problems is to use area-normalized second-derivative spectra. We have found that quantitating the area of overlap between area-normalized spectra provides a reliable, objective method to compare overall spectral similarity. In the current report, we demonstrate this method with selected protein spectra, which were taken from experiments where unfolding was induced by lyophilization or guanidine hydrochloride, and artificial data sets. With this analysis, we document how problems associated with calculation of the correlation coefficient, r, are avoided.

Amides↗
PubMed1995

Infrared spectroscopic studies of lyophilization- and temperature-induced protein aggregation.

Recent studies have clearly demonstrated that Fourier transform IR spectroscopy can be a powerful tool for the study of protein stabilization during freeze-drying and for optimizing approaches to prevent lyophilization-induced protein aggregation. The purpose of the current review is to provide an overview of these topics, as well as an introduction to the study of protein secondary structure with IR spectroscopy. We will start with a general summary of the theories and practices for processing and interpreting protein IR spectra. We will then review the current literature on the use of IR spectroscopy to study protein structure and the effects of stabilizers during lyophilization. Next we will concentrate specifically on protein aggregation. The bulk of the research and the key assignments of spectral features in protein aggregates come from studies of the effects of high and low temperature on proteins. Therefore, we will first consider this topic. Finally, we will summarize the recent theoretical and applied work on lyophilization-induced aggregation.

Chymotrypsin↗
PubMed1991

Functional deficits produced by 3-methylindole-induced olfactory mucosal damage revealed by a simple olfactory learning task.

Methods for assessing functional consequences of olfactory mucosal damage were examined in rats exposed to 3-methylindole (3-MI). Treatment with 3-MI (400 mg/kg) induced severe degeneration of olfactory sensory epithelium followed by regeneration, fibrous adhesions, and osseous remodeling of the nasal passages. At 100 mg/kg, there was mild Bowman's gland hypertrophy while the sensory epithelium remained intact. Rats receiving 3-MI demonstrated a treatment-related deficit in acquiring an olfactory learning task which was not due to altered cognitive abilities, as determined by subsequent testing in a step-through passive avoidance task. The results confirm the conclusion that alterations in functional indices resulted from 3-MI-induced anosmia and demonstrate the utility of simple learning tasks in assessing functional capacity following olfactory epithelial damage in rats.

Animals↗
PubMed1990

Learning and memory deficits in rats following exposure to 3,3'-iminodipropionitrile.

Rats were examined using a learning and memory test battery 4 weeks following exposure to 3,3'-iminodipropionitrile (IDPN). Initial testing revealed deficits in olfactory discrimination and passive avoidance (PA) conditioning. In order to dissociate learning and performance effects, additional tests were conducted. First, to rule out the possibility that IDPN reduced the aversiveness of foot shock, rats were tested in a simple shock sensitivity paradigm. The results indicated no change in shock sensitivity produced by IDPN. Second, to determine if the hyperactivity produced by IDPN was responsible for deficits in conditioning, several additional tests were conducted including (a) repeated-trials active avoidance (AA) and PA conditioning, (b) a PA study which included both a 1- and 24-hr training-testing interval, and (c) long-delay flavor-aversion conditioning. Rats treated with IDPN required more conditioning trials to reach criterion on both AA and PA procedures suggesting that they were capable of performing the required response but acquired those responses at a much slower rate. The deficits in PA conditioning were similar at both the 1- and 24-hr training-testing interval. Finally, the effects of IDPN on flavor-aversion conditioning depended on the delay separating flavor intake and lithium administration during conditioning. Rats treated with IDPN demonstrated robust flavor aversions when trained with a 30-min but not with a 6-hr delay. In summary, the neurotoxic profile of effects produced by IDPN must be expanded to include a prominent cognitive component characterized by protracted deficits in learning and memory capacity. The present experiment illustrates how chemically induced disruption of learning and memory produced by IDPN can be experimentally dissociated from associate neurological symptoms using a simple, routine battery of neurobehavioral tests.

Animals↗
PubMedDate not supplied

Serial electrophysiologic effects of bretylium in man and their correlation with plasma concentrations.

The serial electrophysiologic effects of bretylium were studied in 10 patients undergoing cardiac catheterization before giving the drug, at the end of bretylium tosylate-loading infusion (5 mg/kg/15 min), and after 1 h of intravenous drug maintenance (1.5 mg/min). Mean blood pressure increased during drug loading from 93.6 +/- 10.9 mm Hg to 121.1 +/- 16.2 mm Hg (p less than 0.01, t = 15 min) but returned toward control (100.9 +/- 18.7 mm Hg, p = NS) during drug maintenance (t = 75 min). Small changes in PR, QRS, QTc, AH, and HV intervals during spontaneous and paced rhythms were not significant. Comparison of the control electrophysiologic study with early and late postdrug studies showed no significant changes in corrected sinus node recovery times and in Wenckebach cycle lengths, but small decreases in refractory periods occurred. After drug loading, decreases in mean effective refractory periods of atrium, AV node, and right ventricle of 12, 2, and 16 ms, respectively, occurred (p = NS); during drug maintenance (t = 75 min), these refractory periods had decreased with respect to control by 21, 36, and 13 ms, respectively (p less than 0.05, 0.05, and 0.01). Functional refractory period of the AV node decreased at 75 min by 31 ms (p less than 0.01). The observed shortening of refractory periods contrasts with the direct effects of bretylium in isolated ventricular muscle and Purkinje fibers, which consist of prolongation of effective refractory periods and action potential duration. Thus, indirect (adrenergic) effects may be more important to overall clinical electrophysiologic actions not only acutely during loading but also during at least the first 60-90 min of maintenance therapy, which spans the expected time of initial clinical antifibrillatory response.

Adult↗