The production of freeze-dried liposomes encapsulating drugs is considered a key challenge since the drugs are prone to leakage. The aim of this work was to study the effect of different saccharides on preserving the stability and drug retention capacity of a previously developed liposomal formulation, when subjected to a freeze-drying process. The protective role of trehalose, lactose, glucose, mannitol and sucrose, known for their cryo/lyoprotective effect, was tested by addition of different concentrations to liposomes. Sucrose, in a concentration dependent manner (8:1 sugar:lipids mass ratio) proved to be a suitable cryo/lyoprotectant of these liposomes. Effectively, this saccharide prevents the fusion or/and aggregation of the liposomal formulation, protecting the integrity of the freeze-dried empty liposomes. The liposomal formulation containing sucrose was studied in terms of morphology, concentration, and anticancer drugs retention ability. The study involved two drugs encapsulated in the aqueous core, methotrexate (MTX) and doxorubicin (DOX), and one drug located in the lipid bilayer, tamoxifen (TAM). After the freeze-drying process, liposomes with sucrose encapsulating drugs revealed high physical stability, maintaining their narrow and monodisperse character, however high leakage of the drugs encapsulated in the aqueous core was observed. Otherwise, no significant drug leakage was detected on liposomes containing the TAM, which maintained its biological activity after the freeze-drying process. These findings reveal that sucrose is a good candidate for the cryo/lyoprotection of liposomes with drugs located in the lipid bilayer.

Methotrexate (MTX) is a common drug used to treat rheumatoid arthritis. Due to the excessive side effects, encapsulation of MTX in liposomes is considered an effective delivery system, reducing drug toxicity, while maintaining its efficacy. The ethanol injection method is an interesting technique for liposome production, due to its simplicity, fast implementation, and reproducibility. However, this method occasionally requires the extrusion process, to obtain suitable size distribution, and achieve a low level of MTX encapsulation. Here, we develop a novel pre-concentration method, based on the principles of the ethanol injection, using an initial aqueous volume of 20% and 1:1 ratio of organic:aqueous phase (v/v). The liposomes obtained present small values of size and polydispersity index, without the extrusion process, and a higher MTX encapsulation (efficiency higher than 30%), suitable characteristics for in vivo application. The great potential of MTX to interact at the surface of the lipid bilayer was shown by nuclear magnetic resonance (NMR) studies, revealing mutual interactions between the drug and the main phospholipid via hydrogen bonding. In vivo experiments reveal that liposomes encapsulating MTX significantly increase the biological benefit in arthritic mice. This approach shows a significant advance in MTX therapeutic applications.

In this work a process for the bio-coloration of bacterial cellulose (BC) membranes was developed. Laccase from Myceliophthora thermophila was immobilized onto BC membranes and retained up to 88% of residual activity after immobilization. Four compounds belonging to the flavonoids family were chosen to test the in situ polymerase activity of immobilized laccase. All the flavonoids were successfully polymerized by laccase giving rise to yellow, orange and dark brown oligomers which conferred color to the BC support. The optimal bio-coloration conditions were studied for two of the tested flavonoids, catechol and catechin, by varying the concentration and time of incubation. High color depth and resistance to washing were obtained for both compounds. The highly porous bacterial cellulose material demonstrated great performance as a bio-coloration support, in contrast to other materials cited in literature, like cotton or wool. The process developed is presented as an environmentally friendly alternative for bacterial cellulose bio-coloration and will contribute deeply for the development of new fashionable products within this material.

Sacha inchi oil is rich in essential and non-essential fatty acids and other types of bioactive agents like tocopherols and polyphenolic compounds, which are very well-known antioxidants. In this study, the encapsulation of sacha inchi oil in alginate (AL) and chitosan (CS) nanoparticles was achieved with the assistance of high-intensity ultrasound. Nanoemulsion is the most effective delivery and high stability system for lipophilic bioactive agents. Chitosan and surfactant concentrations were varied to study their effect on particle formulations. Size, zeta-potential, polydispersity, and stability of particles were determined in time to optimize the preparation conditions. Sacha inchi oil encapsulated in AL-CS nanoparticles showed a higher loading efficiency and stability for short and long periods compared with other vegetable oils such as olive and soybean. Also, because of the types of tocopherols present in sacha inchi oil (γ- and δ-tocopherols), a much higher antioxidant activity (95% of radical reduction in 15 min) was found in comparison with nanocapsules with olive oil, which contain α-tocopherols. The particles showed high efficiency of protein loading at high concentration of bovine serum albumin (BSA) and a low rate of leaching profiles in various testing media like simulated gastric and intestinal fluids with/without enzymes, that is, pepsin 0.1% (w/v) and pancreatin 0.1% (w/v), respectively.