Development and optimization of a one step process for the production and sterilization of liposomes using supercritical CO2.

Liposomes are very interesting drug delivery systems for pharmaceutical and therapeutic purposes. However, liposome sterilization as well as their industrial manufacturing remain challenging. Supercritical carbon dioxide is an innovative technology that can potentially overcome these limitations. The aim of this study was to optimize a one-step process for producing and sterilizing liposomes using supercritical CO2. For this purpose, a design of experiment was conducted. The analysis of the experimental design showed that the temperature is the most influential parameter to achieve the sterility assurance level (SAL) required for liposomes (≤10-6). Optimal conditions (80 °C, 240 bar, 30 min) were identified to obtain the fixed critical quality attributes of liposomes. The conditions for preparing and sterilizing empty liposomes of various compositions, as well as liposomes containing the poorly water-soluble drug budesonide, were validated. The results indicate that the liposomes have appropriate physicochemical characteristics for drug delivery, with a size of 200 nm or less and a PdI of 0.35 or less. Additionally, all liposome formulations demonstrated the required SAL and sterility at concentrations of 5 and 45 mM, with high encapsulation efficiency.

Use of supercritical CO2 for the sterilization of liposomes: Study of the influence of sterilization conditions on the chemical and physical stability of phospholipids and liposomes.

The effects of four potential supercritical carbon dioxide (ScCO2) sterilization conditions on the chemical stability of 9 phospholipids and on the physicochemical characteristics of liposomes consisting of stable phospholipids, as well as their sterilization efficiency were evaluated. These conditions were : C1 (ScCO2/70 °C/150 bar/240 min), C2 (ScCO2/0.25 % water/ 0.15% H2O2/ 0.5% acetic anhydride/38° C/85 bar/45 min), C3 (ScCO2/0.08 % peracetic acid/35° C/104 bar/180 min) and C4 (ScCO2/200 ppm H2O2/40 °C/270 bar/90 min). The results showed for phospholipids, a significant increase in hydrolysis products of 3.77 to 14.50 % and an increase in oxidation index of 6.10 to 430.50 % with unsaturated phospholipids for all tested conditions while with saturated phospholipids, no significant degradation was observed. Concerning the liposome formulation, no change in dispersion color and no phospholipid degradation were observed. However, a decrease in liposome size from 126.90 nm to 111.80 nm, 96.27 nm, 99.60 nm and 109.13 nm and an increase in the PdI from 0.208 to 0.271, 0.233, 0.285, and 0.298 were found with conditions C1, C2, C3 and C4 respectively. For the sterilization efficiency, conditions C1, C2 and C3 achieved the required sterility assurance level (SAL) of 10-6 for liposomes.

An innovative one step green supercritical CO2 process for the production of liposomes co-encapsulating both a hydrophobic and a hydrophilic compound for pulmonary administration.

Budesonide and salbutamol-loaded liposomes were prepared using an innovative one step supercritical CO2 method without any use of organic solvents. Liposomes composed of soybean phosphatidylcholine, cholesterol and PEGylated lipid (65/30/5% (m/m)) were produced with a size less than 200 nm, a PdI within the range of 0.3 and 0.35 and encapsulation efficiency for budesonide and salbutamol reaching to 94% and 40% respectively. The physical stability of the formulation was improved by optimizing a dry form by freeze-drying with trehalose in a 20:1 (trehalose:lipid) ratio and an increase in the percentage of PEGylated lipid from 5% to 15%. This dry form stored at 4 °C maintains 90-110% of the initial concentration of active compounds. The concentration of budesonide and salbutamol after 15 weeks was 522.92 ± 73.01 µg/mL and 144.86 ± 31.22 µg/mL respectively. These concentrations are close to the concentrations of these molecules in the pharmaceutical products Pulmicort® (500 µg/mL of budesonide) and Ventolin® (100 µg/dose). The formulation tested on lung cells, allows a cell viability of 71 ± 6%, which is not significantly different from untreated cells.

Sterilization methods of liposomes: Drawbacks of conventional methods and perspectives.

Liposomes are targeted drug delivery systems that are of great pharmaceutical and therapeutic interest. Parenteral route is the main way used for liposome administration. In this case, their sterility is a requirement. However, due to the particular sensitivity of liposomes and their tendency to physicochemical alterations, their sterilization remains a real challenge. Conventional sterilization methods such as heat, ethylene oxide, ultraviolet and gamma irradiations are considered as unsuitable for liposome sterilization and the recommended methods for obtaining sterility of liposomes are filtration and aseptic manufacturing. Unfortunately, these recommended methods are not without limitations. This review outlines the difficulties associated with the use of these different classical methods for obtaining liposome sterility. The effects on liposome physicochemical and biopharmaceutical characteristics as well as efficacy, toxicity and practical problems of these sterilization techniques have been discussed. The search for an alternative method being therefore necessary, the applicability of supercritical carbon dioxide (ScCO2) technology, which is nowadays a promising strategy for the sterilization of sensitive products such as liposomes, is also examined. It appears from this analysis that ScCO2 could effectively be an interesting alternative to achieve sterility of liposomes, but for this, sterilization assays including challenge tests and optimization studies are needed.