INVESTIGATION OF THE STABILITY OF THERMOSENSITIVE LIPOSOMES PREPARED BY DUAL CENTRIFUGATION

In the last decades, liposomes have been widely exploited as drug delivery systems that, if properly formulated, allow the encapsulation, protection and delivery of a drug substance at its site of action. Thermosensitive liposomes are promising vehicles designed to release their content only at moderate hyperthermia, therefore increasing the therapeutical effectiveness of the encapsulated drug substances when the site of action is heated up by external means. Despite these advantages, the formulation of stable thermosensitive liposomes remains a matter of concern in the setting of drug delivery. Focusing on the newly developed and very promising thermosensitive liposomes (DPPC/DSPC/DPPG2 thermosensitive liposomes (DPPG2-TSL)), one (out of some more) important aspect here is the rather fast hydrolysis of the novel lipid DPPG2 as main constituent of the new DPPG2-TSL. DPPG2-TLS can be prepared as unilamellar but also as multilamellar liposomes by using dual centrifugation (DC), performing the homogenization process either with lower or with higher lipid concentrations, respectively. Thus, the aim of this study was to gain a better understanding of the process of lipid hydrolysis occurring during the manufacturing process at high and low lipid concentrations as well as during storage and to determine the most stable DPPG2-TSL formulation for a possible clinical application. After establishing a HPTLC-method to determine the hydrolysis-products, lyso- DPPG2 and free fatty acid, hydrolysis of DPPG2-TSL during preparation was investigated and compared to lamellarity. Furthermore, the impact of polyalcohols and pH variations on the hydrolytic stability of the different DPPG2-TSL formulations was tested as well. Moreover, this work evaluates the effect of the lyophilization on the hydrolytic stability of DPPG2- TSL. Further analysis were conducted to test different storage conditions on not lyophilized samples for a possible promising clinical application. It was found that DPPG2 shows significantly faster hydrolysis during DC-homogenization than pure phosphatidylcholines, but it was surprising that hydrolysis was even more pronounced when liposomes were made of mixtures of DPPG2 and phosphatidylcholines instead of pure DPPG2. However, it could be shown that DPPG2 hydrolysis is strongly reduced when DC-homogenization was performed with very high concentrations of lipid which results in small multilamellar vesicles (SMV). Furthermore, a very surprising finding was that lyophilization resulted in a higher DPPG2- hydrolysis than simple storage of the liposomes at 4°C. Based on the findings of this study, a hypothesis was drawn which explains the very unusual sensitivity of DPPG2-TSL to hydrolysis during manufacturing (homogenization). This initial study gained the knowledge about the stability of DC-prepared DPPG2-TSL formulations and showed that it is possible to prepare DPPG2-TSL without significant hydrolysis by dual centrifugation using high lipid concentrations, resulting in SMVs