Operative parameters optimization production of liposomes for the encapsulation of hydrophilic compounds using a new supercritical process

Liposomes are spherical vesicles formed by a inner aqueous core and a double lipidic layer around it. Conventional techniques for the production of liposomes are characterized by several drawbacks, like the production of micrometric vesicles, a difficult control of the Particle Size Distribution (PSD) and low encapsulation efficiencies (EE) of hydrophilic compounds. Many supercritical semi-continuous techniques were proposed in literature. They are successful in the intent of producing liposomes of smaller diameter, but the EE of hydrophilic compounds and the reproducibility are still a challenge. For this reason, it was recently proposed a new supercritical process whose aim is to invert the steps of production of liposomes, by first creating water droplets and then to fast surround them by phospholipids. We discovered that the high diffusion coefficient of phospholipids in supercritical carbon dioxide allows a fast coverage of water droplets preserving the drug content into the liposome core. In this work, hydrophilic compounds were encapsulated in the vesicles produced using SuperLip, such as Fluorescein, Bovine Serum Albumin (BSA) and Ampicillin, obtaining monodispersed spherical vesicles with a mean size from 100 to 300 nm. Operative parameters like water flow rate and lipid to water mass ratio were optimized. The EEs were evaluated with UV-Vis spectroscopy according to methods reported in literature, and obtaining high values up to 99 % for the three investigated compounds

Analysis of dissolved-gas atomization: supercritical CO2 dissolved in water

Supercritical dissolved-gas atomization is an atomization process in which carbon dioxide at temperature and pressure above its critical point is used as the atomizing gas. The spray characteristics in terms of droplets size and distribution have been experimentally studied using a laser diffraction method based on a Malvern apparatus. The main parameter that influences the droplets size is the gas-to-liquid mass ratio (GLR); the injection pressure in the range of 7.4-13 MPa has a minor effect. Upon variation of the GLR from 0.5 to 3, the droplet mean diameter changes from about 8.0 to 2.0 μm; very narrow droplet size distributions are also produced. From the point of view of the atomization mechanism, the mean droplet diameter is mainly influenced by the sudden release of the gas dissolved in the liquid. The overall analysis of the experimental data confirms that dissolved-gas atomization allows for the formation of micrometric droplets that can produce precipitates with controlled sizes and distributions that are useful in several fine-particles production processes

Liposomes: From Bangham to Supercritical Fluids

Liposomes are spherical vesicles made up of an aqueous core surrounded by phospholipids. These delivery systems (DS) are largely employed as drug carriers in several industrial fields, such as pharmaceutical and nutraceutical fields. The aim of this short review is to provide a fast overview on the main fundamentals of liposomes, thought as a compact guide for researchers and students that want to approach this topic for the first time. The mini-review will focus on the definitions, production methods and characterization protocols of the liposomes produced, making a critical comparison of the main conventional and supercritical based manufacturing methods available. The literature was analyzed deeply from the first works by Dr. Bangham in 1965 to the most recent supercritical fluid applications. The advantages and disadvantages of conventional and high-pressure processes will be described in terms of solvent elimination, production at the nanometric (50–300 nm) and micrometric level (1–100 μm) and encapsulation efficiency (20–90%). The first proposed methods were characterized by a low encapsulation efficiency (20–40%), resulting in drug loss, a high solvent residue and high operating cost. The repeatability of conventional processes was also low, due to the prevalent batch mode. Supercritical-assisted methods were developed in semi-continuous layouts, resulting in an easy process scale-up, better control of liposome dimensions (polydispersity index, PDI) and also higher encapsulation efficiencies (up to 90%)

Towards a Quantitative Modeling of the Synthesis of the Pectate Lyases, Essential Virulence Factors in Dickeya Dadantii

A dynamic mathematical model has been developed and validated to describe the synthesis of pectate lyases (Pels), the major virulence factors in Dickeya dadantii. This work focuses on the simultaneous modeling of the metabolic degradation of pectin by Pel enzymes and the genetic regulation of pel genes by 2-keto-3-deoxygluconate (KDG), a catabolite product of pectin which inactivates KdgR, one of the main repressors of pel genes. This modeling scheme takes into account the fact that the system is composed of two time-varying compartments: the extracellular medium, where Pel enzymes cleave pectin into oligomers, and the bacterial cytoplasm where, after internalization, oligomers are converted to KDG. Using the quasi-stationary state approximations, the model consists of some nonlinear differential equations for which most of the parameters could be estimated from the literature or from independent experiments. The few remaining unknown parameters were obtained by fitting the model equations against a set of Pel activity data. Model predictions were verified by measuring the time courses of bacterial growth, Pel production, pel mRNA accumulation and pectin consumption under various growth conditions. This work reveals that pectin is almost totally consumed before the burst of Pel production. This paradoxical behaviour can be interpreted as an evolutionary strategy to control the diffusion process so that as soon as a small amount of pectin is detected by the bacteria in its surroundings it anticipates more pectin to come. The model also predicts the possibility of bistable steady states in the presence of constant pectin compounds.Comment: 21 pages, 11 figures, Journal of Biological Chemistry (In press