A physicochemical assessment of the thermal stability of dextrin-colistin conjugates.

Attachment of polysaccharide carriers is increasingly being used to achieve precision delivery and improved effectiveness of protein and peptide drugs. Although it is clear that their clinical effectiveness relies on the purity and integrity of the conjugate in storage, as well as following administration, instability of polysaccharide-based conjugates can reduce the protective efficacy of the polymer, which may adversely affect the bioactive's potency. As a model, these studies used dextrin-colistin conjugates, with varying degrees of polymer modification (1, 2.5 and 7.5 mol% succinoylation) to assess the effect of storage temperature (- 20, 4, 21 and 37 °C) and duration (up to 12 months) on saccharide and colistin release and antimicrobial activity. Estimation of the proportion of saccharide release (by comparison of area under the curve from size exclusion chromatograms) was more pronounced at higher temperatures (up to 3 and 35% at - 20 °C and 37 °C, respectively after 12 months), however, repeated freeze-thaw did not produce any measurable release of saccharides, while addition of amylase (20, 100, 500 IU/L) caused rapid release of saccharides (> 70% total within 24 h). At all temperatures, conjugates containing the lowest degree of succinoylation released the highest proportion of free colistin, which increased with storage temperature, however no trend in saccharide release was observed. Despite the clear physical effects of prolonged storage, antimicrobial activity of all samples was only altered after storage at 37 °C for 12 months (> threefold decreased activity). These results demonstrate significant release of saccharides from dextrin-colistin conjugates during prolonged storage in buffered solution, especially at elevated temperature, which, in most cases, did not affect antimicrobial activity. These findings provide vital information about the structure-activity relationship of dextrin-colistin conjugates, prior to full-scale commercial development, which can subsequently be applied to other polysaccharide-protein and -peptide conjugates.

A linkage between SmeIJK efflux pump, cell envelope integrity, and σE-mediated envelope stress response in Stenotrophomonas maltophilia.

Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of Stenotrophomonas maltophilia, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σE-mediated envelope stress response (ESR) of S. maltophilia was assessed. SmeIJK was involved in the intrinsic resistance of S. maltophilia KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the smeIJK deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σE-mediated ESR. Moreover, the expression of smeIJK was further induced by sub-lethal concentrations of MDAs or surfactants in an σE-dependent manner. These data collectively suggested an alternative physiological role of smeIJK in cell envelope integrity maintenance and σE-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting S. maltophilia from the envelope stress, smeIJK is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σE-mediated ESR in S. maltophilia.

Polymyxin E sulfate-loaded liposome for intravenous use: preparation, lyophilization, and toxicity assessment in vivo.

Polymyxin E sulfate, a hydrophilic drug with high tissue toxicity, was formulated into a stable liposome to reduce its in vivo toxicity. The liposome was prepared using both a reverse phase evaporation and freezing-thawing method. The encapsulation efficiency was determined by high-performance liquid chromatography. The influence of the freezing-thawing process on the liposome's stability was evaluated using a centrifugation test. The dialysis method was employed to investigate the in vitro release profile of the drug-loaded liposome. The toxicity of the polymyxin E sulfate-loaded liposome was compared with the polymyxin E sulfate solution in Kunming mice by intravenous administration. A freeze-drying (lyophilization) technique was utilized to prepare the proliposome. A cryoprotectant formulation was optimized by the evaluation of the particle diameter and encapsulation efficiency of the redispersed proliposomes, while the influence of the concentration and method of the addition of the cryoprotectant on the protective effect was investigated as well. It was found that the stability and encapsulation efficiency of the liposome could be improved by the freezing-thawing process. The delayed drug release profile of the drug-loaded liposome was observed in vitro, and a comparison with the polymyxin E sulfate solution revealed that the in vivo toxicity of the polymyxin E sulfate-loaded liposome was significantly reduced. Sucrose and mannitol at a weight ratio of 1:0.8:0.6 (phospholipids:mannitol:sucrose) added inside displayed the greatest protective effect on the polymyxin E sulfate liposome during freeze-drying. The rehydrated proliposomes with optimized cryoprotectants produced an acceptable particle diameter (204.1 nm) and a satisfactory encapsulation efficiency percentage (51.48%), thus demonstrating a practical method for preparation of polymyxin E sulfate-loaded proliposome. A combination of the reverse phase evaporation and freezing-thawing methods was found to be suitable for the preparation of hydrophilic drug-loaded liposome. The toxicity of polymyxin E sulfate was reduced by its encapsulation in a liposome, thus constituting a promising drug delivery system suitable for further development. The method of addition had an insignificant effect on the appearance of the product but obviously influenced the particle diameter and encapsulation efficiency. The inside addition method produced a greater protective effect in this study.