We developed lipid-like ionic liquids, containing 2-mercaptoimidazolium and 2-mercaptothiazolinium headgroups tethered to two long saturated alkyl chains, as carriers for in vitro delivery of plasmid HEK DNA into 293T cells. We employed a combination of modular design, synthesis, X-ray analysis, and computational modeling to rationalize the self-assembly and desired physicochemical and biological properties. The results suggest that thioamide-derived ionic liquids may serve as a modular platform for lipid-mediated gene delivery. This work represents a step toward understanding the structure-function relationships of these amphiphiles with long-range ordering and offering insight into design principles for synthetic vectors based on self-assembly behavior.

Lipid droplets (LDs) are important intracellular organelles that store energy in the form of neutral lipids, mainly triglycerides. Lipophagy, a process in which lysosomal enzymes are used to break down LDs, is an essential pathway for energy release. Obesity is thought to be associated with downregulation of lysosomal enzymes and impaired autophagy, which contributes to the development of non-alcoholic fatty liver disease. In this study, the effects of palmitic acid (PA) and oleic acid (OA) on LD metabolism with a particular focus on the autophagy-lysosomal pathway were investigated using mouse liver samples and in vitro hepatocyte models. Results showed a different progression of non-alcoholic steatohepatitis (NASH) with specific fat-enriched diets. While a diet enriched with monounsaturated fatty acids (MUFA) primarily caused hepatic steatosis in the mice, a diet enriched with saturated fatty acids (SFA) additionally led to lobular inflammation, liver cell ballooning and fibrosis besides steatosis. An increase in perilipin 2 (PLIN2), a lipid membrane protein, was observed with both diets. Both SFA- and MUFA-enriched diets increased expression of ATGL lipase as well as expression and activity of lysosomal enzymes, suggesting a compensatory mechanism in liver tissue to counteract the increased lipid content. While autophagy marker proteins were increased in liver tissue in response to both lipid-enriched diets, only the SFA diet resulted in increased expression of Rubicon, a negative regulator of autophagosome-lysosome fusion, particularly in the older mice. Analysis of liver tissue from mice fed MUFA- and SFA-enriched diets indicated a differential autophagy response to the specific lipid diets. Since the murine diets did not contain only one specific fatty acid, in vitro experiments were subsequently performed to investigate the direct effects of SFA-PA and MUFA-OA on lipid droplet metabolism and autophagy in liver cell types. PA and OA were used in ethanol and in bovine serum albumin (BSA) complexes to investigate their influence on lipid droplet metabolism and autophagy in different liver cell types. Cells treated with fatty acids in BSA complex showed higher intracellular fatty acid concentrations than those in ethanol, which correlated with decreased cell survival, increased LD accumulation and PLIN2 expression. OA enhanced lipid droplet accumulation and PLIN2 expression while preserving lysosomal integrity and autophagy function. Conversely, PA inhibited lysosomal enzyme function and decreased autophagy flux. The latter could possibly be caused by reduced autophagosome-lysosome fusion, as indicated by the upregulation of Rubicon and reduced co-localization of autophagosomes with lipid droplets and lysosomes. In summary, our results provide valuable insights into the LD metabolism influenced by fatty acids using liver tissue and cell models. In vitro studies showed different effects of PA and OA on LD metabolism. While OA increased LD accumulation and maintained autophagy-lysosome function, PA impaired autophagosome-lysosome-fusion, resulting in decreased autophagic flux. Alteration of autophagy by PA may play a role in the progression of NASH and subsequent liver fibrosis.

Purpose Systematic comparison of analysis methods of clinical microdialysis data for impact on target-site drug exposure and response. Methods 39 individuals received a 500 mg levofloxacin short-term infusion followed by 24-h dense sampling in plasma and microdialysate collection in interstitial space fluid (ISF). ISF concentrations were leveraged using non-compartmental (NCA) and compartmental analysis (CA) via (ii) relative recovery correction at midpoint of the collection interval (midpoint-NCA, midpoint-CA) and (ii) dialysate-based integrals of time (integral-CA). Exposure and adequacy of community-acquired pneumonia (CAP) therapy via pharmacokinetic/pharmacodynamic target-attainment (PTA) analysis were compared between approaches. Results Individual AUC(ISF) estimates strongly varied for midpoint-NCA and midpoint-CA (>= 52.3%CV) versus integral-CA (<= 32.9%CV) owing to separation of variability in PK parameters (midpoint-CA = 46.5%-143%CVPK, integral-CA = 26.4%-72.6%CVPK) from recovery-related variability only in integral-CA (41.0%-50.3%CVrecovery). This also led to increased variability of AUC(plasma) for midpoint-CA (56.0%CV) versus midpoint-NCA and integral-CA (<= 33.0%CV), and inaccuracy of predictive model performance of midpoint-CA in plasma (visual predictive check). PTA analysis translated into 33% of evaluated patient cases being at risk of incorrectly rejecting recommended dosing regimens at CAP-related epidemiological cut-off values. Conclusions Integral-CA proved most appropriate to characterise clinical pharmacokinetics- and microdialysis-related variability. Employing this knowledge will improve the understanding of drug target-site PK for therapeutic decision-making.