Electrospun Amphiphilic Nanofibers as Templates for In Situ Preparation of Chloramphenicol-Loaded Liposomes

The hydration of phospholipids, electrospun into polymeric nanofibers and used as templates for liposome formation, offers pharmaceutical advantages as it avoids the storage of liposomes as aqueous dispersions. The objective of the present study was to electrospin and characterize amphiphilic nanofibers as templates for the preparation of antibiotic-loaded liposomes and compare this method with the conventional film-hydration method followed by extrusion. The comparison was based on particle size, encapsulation efficiency and drug-release behavior. Chloramphenicol (CAM) was used at different concentrations as a model antibacterial drug. Phosphatidylcoline (PC) with polyvinylpyrrolidone (PVP), using ethanol as a solvent, was found to be successful in fabricating the amphiphilic composite drug-loaded nanofibers as well as liposomes with both methods. The characterization of the nanofiber templates revealed that fiber diameter did not affect the liposome size. According to the optical microscopy results, the immediate hydration of phospholipids deposited on the amphiphilic nanofibers occurred within a few seconds, resulting in the formation of liposomes in water dispersions. The liposomes appeared to aggregate more readily in the concentrated than in the diluted solutions. The drug encapsulation efficiency for the fiber-hydrated liposomes varied between 14.9 and 28.1% and, for film-hydrated liposomes, between 22.0 and 77.1%, depending on the CAM concentrations and additional extrusion steps. The nanofiber hydration method was faster, as less steps were required for the in-situ liposome preparation than in the film-hydration method. The liposomes obtained using nanofiber hydration were smaller and more homogeneous than the conventional liposomes, but less drug was encapsulated.

3D Printed Integrated Multi-Layer Microfluidic Chips for Ultra-High Volumetric Throughput Nanoliposome Preparation

Although microfluidic approaches for liposomes preparation have been developed, fabricating microfluidic devices remains expensive and time-consuming. Also, owing to the traditional layout of microchannels, the volumetric throughput of microfluidics has been greatly limited. Herein an ultra-high volumetric throughput nanoliposome preparation method using 3D printed microfluidic chips is presented. A high-resolution projection micro stereolithography (PμSL) 3D printer is applied to produce microfluidic chips with critical dimensions of 400 µm. The microchannels of the microfluidic chip adopt a three-layer layout, achieving the total flow rate (TFR) up to 474 ml min−1, which is remarkably higher than those in the reported literature. The liposome size can be as small as 80 nm. The state of flows in microchannels and the effect of turbulence on liposome formation are explored. The experimental results demonstrate that the 3D printed integrated microfluidic chip enables ultra-high volumetric throughput nanoliposome preparation and can control size efficiently, which has great potential in targeting drug delivery systems.

Application of Asymmetrical Flow Field-Flow Fractionation for Characterizing the Size and Drug Release Kinetics of Theranostic Lipid Nanovesicles

Liposome size and in vitro release of the active substance belong to critical quality attributes of liposomal carriers. Here, we apply asymmetric flow field-flow fractionation (AF4) to characterize theranostic liposomes prepared by thin lipid film hydration/extrusion or microfluidics. The vesicles’ size was derived from multi-angle laser light scattering following fractionation (AF4) and compared to sizes derived from dynamic light scattering measurements. Additionally, we adapted a previously developed AF4 method to study zinc phthalocyanine (ZnPc) release/transfer from theranostic liposomes. To this end, theranostic liposomes were incubated with large acceptor liposomes serving as a sink (mimicking biological sinks) and were subsequently separated by AF4. During incubation, ZnPc was transferred from donor to acceptor fraction until reaching equilibrium. The process followed first-order kinetics with half-lives between 119.5–277.3 min, depending on the formulation. The release mechanism was postulated to represent a combination of Fickian diffusion and liposome relaxation. The rate constant of the transfer was proportional to the liposome size and inversely proportional to the ZnPc/POPC molar ratio. Our results confirm the usefulness of AF4 based method to study in vitro release/transfer of lipophilic payload, which may be useful to estimate the unwanted loss of drug from the liposomal carrier in vivo.

Comparative Evaluation of Artificial Neural Networks and Data Analysis in Predicting Liposome Size in a Periodic Disturbance Micromixer

Artificial neural networks (ANN) and data analysis (DA) are powerful tools for supporting decision-making. They are employed in diverse fields, and one of them is nanotechnology; for example, in predicting silver nanoparticles size. To our knowledge, we are the first to use ANN to predict liposome size (LZ). Liposomes are lipid nanoparticles used in different biomedical applications that can be produced in Dean-Forces-based microdevices such as the Periodic Disturbance Micromixer (PDM). In this work, ANN and DA techniques are used to build a LZ prediction model by using the most relevant variables in a PDM, the Flow Rate Radio (FRR), and the Total Flow Rate (TFR), and the temperature, solvents, and concentrations were kept constant. The ANN was designed in MATLAB and fed data from 60 experiments with 70% training, 15% validation, and 15% testing. For DA, a regression analysis was used. The model was evaluated; it showed a 0.98147 correlation coefficient for training and 0.97247 in total data compared with 0.882 obtained by DA.

UV Properties and Loading into Liposomes of Quinoline Derivatives

The scientific relevance of quinolines is strictly linked to the fine-tuning of their features by functionalizing the heterocyclic core. Consequently, the compounds of this class are very versatile and can be used as possible drugs for a lot of medical applications. In this work, the inclusion of eight synthetic quinoline derivatives in liposomes formulated with different lipids was investigated in terms of the encapsulation efficiency and to highlight the effect on the liposome size distribution and thermotropic behavior. Excellent encapsulation was accomplished with all the quinoline/phospholipid combinations. Differences in the interactions at the molecular level, dependent on the quinoline molecular scaffolds and lipid structure, were observed, which could significantly bias the interaction with the drug and its release in pharmaceutical applications. Experiments in combination with computational studies demonstrated that the UV absorption of quinolines with expanded conjugation could be affected by the environment polarity. This was probably due to a solvent-dependent ability of these quinolines to stack into aggregates, which could also occur upon inclusion into the lipid bilayer.

Comparative Evaluation of Artificial Neural Networks and Data Analysis in Predicting Liposomes’ Size in a Periodic Disturbance Micromixer

Artificial Neural Networks (ANN) and Data analysis are powerful tools used for supporting decision-making. They have been employed in diverse fields and one of them is nanotechnology used, for example, in predicting particles size. Liposomes are nanoparticles used in different biomedical applications that can be produced in Dean Forces-based Periodic Disturbance Micromixers (PDM). In this work, ANN and data analysis techniques are used to build a liposome size prediction model by using the most relevant variables in a PDM, i.e., Flow Rate Radio (FRR) and Total Flow Rate (TFR). The ANN was designed in MATLAB and fed data from 60 experiments, which were 70% training, 15% validation and 15% testing. For data analysis, regression analysis was used. The model was evaluated; it showed 98.147% of regression number for training and 97.247% in total data compared with 78.89% regression number obtained by data analysis. These results demonstrate that liposomes’ size can be better predicted by ANN with just FRR and TFR as inputs, compared with data analysis techniques when the temperature, solvents, and concentrations are kept constant.

Aggregation-related quenching of LHCII in liposomes revealed by single-molecule spectroscopy

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII-LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Continuous-Flow Production of Liposomes with a Millireactor under Varying Fluidic Conditions

Continuous-flow production of liposomes using microfluidic reactors has demonstrated advantages compared to batch methods, including greater control over liposome size and size distribution and reduced reliance on post-production processing steps. However, the use of microfluidic technology for the production of nanoscale vesicular systems (such as liposomes) has not been fully translated to industrial scale yet. This may be due to limitations of microfluidic-based reactors, such as low production rates, limited lifetimes, and high manufacturing costs. In this study, we investigated the potential of millimeter-scale flow reactors (or millireactors) with a serpentine-like architecture, as a scalable and cost-effective route to the production of nanoscale liposomes. The effects on liposome size of varying inlet flow rates, lipid type and concentration, storage conditions, and temperature were investigated. Liposome size (i.e., mean diameter) and size dispersity were characterised by dynamic light scattering (DLS); z-potential measurements and TEM imaging were also carried out on selected liposome batches. It was found that the lipid type and concentration, together with the inlet flow settings, had significant effects on the properties of the resultant liposome dispersion. Notably, the millifluidic reactor was able to generate liposomes with size and dispersity ranging from 54 to 272 nm, and from 0.04 to 0.52 respectively, at operating flow rates between 1 and 10 mL/min. Moreover, when compared to a batch ethanol-injection method, the millireactor generated liposomes with a more therapeutically relevant size and size dispersity.

Alpha Tocopherol Loaded in Liposome: Preparation, Optimization, Characterization and Sperm Motility Protection

Background: Alpha-tocopherol is a potent antioxidant involved in sperm protection particularly during cryopreservation. However, its poor solubility limits the optimal protection in aqueous solutions. Objective: The aim of this study was to enhance the solubility of α-tocopherol by the use of liposomes. Methods: The experimental approach consisted to load vitamin E in liposomes prepared by ethanol injection method and the optimization carried out by an experimental design. The optimum solution was characterized by high performance liquid chromatography and scanning electron microscope. Finely, the impact on sperm motility protection was studied by the freezing technic of bovine sperm. Results: The optimum solution was obtained when using 10.9 mg/ml of phospholipids, 1.7 mg/ml of cholesterol and 2 mg/ml of vitamin E. The liposome size was 99.86 nm, providing 78.47% of loaded efficiency. The results showed also a significant positive impact on sperm motility after hours of preservation. Conclusion: In conclusion, the current results showed the interest of liposome preparation as an alternative to enhance vitamin E solubility and to protect spermatozoa during cryopreservation.