# Steering the Clinical Translation of Delivery Systems for Drugs and Health Products
## 3 Poster Presentations
### Is It Possible to Protect Liposomes Prepared by Ethanol Injection Upon Freeze-Drying  
[PERSON] *, [PERSON], [PERSON], [PERSON] and [PERSON]  
Department Pharmaceutical Sciences, Universita degli Studi di Milano, via G. Colombo 71, 20133 Milan, Italy  
* Correspondence: [EMAIL_ADDRESS]  
Ethanol injection is an attractive technique for scaling-up liposome production. It is easily feasible at laboratory scale without sophisticated equipment and the method can be implemented at industrial scale by using crossflow or microfluidic devices, appositely developed [85]. However, the purification from residual solvent is often required. lyophilization of the liposomal dispersion would avoid this step, increasing concomitantly the shelf-life of the product [86]. Nevertheless, the evaporation of ethanol, which does not freeze completely, may affect the quality of the freeze-dried product and therefore the reconstitution of the liposome dispersion.  
This work aims to study the influence of the residual ethanol content on the feasibility of freeze-drying hydro-alcoholic liposomal dispersions. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (70:30 mol/mol) sizing 130 nm were prepared by ethanol injection and processed to have different final ethanol contents, namely 0.1, 1 and 6% v/v. Liposomes prepared by thin film hydration method were used as a negative control. The possible interactions with trehalose or saccharose (5:1 molar ratio), poly(vinyl pyrrolone) K12 (PVP) (0.50 or 0.75%) and the binary mixtures were experimentally and computationally investigated.  
All excipients were compatible with liposomes, even if PVP caused the formation of large aggregates after thawing and trehalose proved to be a better cryoprotectant than sucrose. This result agreed with the molecular dynamic simulations since both the free energy difference calculations and dipole alignment demonstrated that interactions with a DPPC bilayer were stronger for trehalose than sucrose.  
Based on compatibility results, only hydro-alcoholic liposomal dispersions containing trehalose, PVP at 0.5% w/v or combination thereof were subject to thawing. The liposome characteristics were not altered upon freezing in agreement with the microregion entrapment theory. Indeed, upon freezing the ethanol remained uniformly entrapped as a liquid in the solid microregions allowing liposomes to reorganize without undergoing damages caused by ice crystals or cryo-concentration. Thus, the possible effect of protectants upon thawing was deepened on liposomes without ethanol. Trehalose and PVP at 0.5% m/v in mixture permitted to preserve liposome size distribution upon thawing.  
After lyophilization, the moisture and the ethanol content were lower than 1% and negligible, respectively. However, the cake appearance was dependent of the ethanol content in the original dispersion. Up to 1%, the dried formulation was \"blown out\" of the vial (\"product ejection\") because of the poor cohesion within the cake and solvent vapor pressure upon sublimation.  
Upon reconstitution of freeze-dried products in the presence of a single protectant, the formation of too dispersed aggregates prevented DLS analysis. At ethanol contents lower that 1%, cakes presented an acceptable consistency and the reconstitution in water occurred within 30 min. However, liposomes freeze-dried in the presence of a single protectant showed an increased size (DH = 550 nm). Size comparable to that before drying (DH = 232 \(\pm\) 15 nm) were obtained instead after reconstitution of formulation obtained using the binary mixture of excipients.  
In conclusion, liposomes prepared by ethanol injection must be subjected unavoidably to mild processes of solvent evaporation before freeze-drying. A small residual ethanol content is allowed when a combination of trehalose and PVP is added to the formulation to preserve liposome integrity.