Protecting Effect of Threalose and PVP in Freeze-Drying Liposomes Prepared by Ethanol Injection
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Data di Pubblicazione:
2018
Citazione:
Protecting Effect of Threalose and PVP in Freeze-Drying Liposomes Prepared by Ethanol Injection / F. Selmin, S. Franzè, U. Musazzi, P. Minghetti, F. Cilurzo. ((Intervento presentato al convegno AAPS PharmSci 360 tenutosi a Washington DC nel 2018.
Abstract:
Purpose: The use of ethanol injection to produce liposomes is rising an increasing interest since it is a scalable method, that allows to obtain small unilamellar vesicles in one step, overcoming further downsizing steps. On the other hand, the presence of a co-solvent which does not freeze completely, causes several issues during the lyophilization step. Hence, this study investigated the influence of residual ethanol content, the type and the concentration of protectants on liposome stability to assure the desired characteristics of the final freeze-dried product. Methods: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)–cholesterol liposomes were prepared by ethanol injection with a 6% v/v ethanol content. The ethanol was removed by rotary evaporation under reduced pressure to obtain the final concentration of 1 or 0.1% as determined by gas chromatography. Liposomes were also prepared without ethanol by thin film hydration method. The compatibility and potential protective effect of threalose and/or poly(vinyl pyrrolidone) K12 (PVP), was assessed by DLS before and after thawing. The freeze drying was performed by an Epsilon 2-6 LC plus freeze dyer (Martin Christ, D). The experimental conditions were defined according to DSC data. The residual moisture content of lyophilized formulations was determined by Karl Fischer titration. Afterwards, freeze-dried products were reconstituted in water to their original volume under gentle shaking at 100 rpm and 25 °C for 30 min. Size and -potential were evaluated in samples without sign of visually detectable aggregates. Results: Liposomes prepared by ethanol injection exhibited a size around 130 nm and a narrow size distribution (PDI0.15). The compatibility study revealed that threalose in molar ratio 5:1 to DPPC did not alter the size distribution of liposomes, but determined a slight decrease of -potential from -8.0±1.1 to -12.7±0.9 mV due to its absorption on vesicle surface. PVP at concentration up to 1 % caused the -potential to shift towards less negative values with a concomitant increase in size and dispersity of liposomes. This behaviour was evidenced after adding not only PVP as such, but also in combination with threalose. Hence only formulations containing threalose, PVP at 0.5% m/v or combination thereof were freeze-thawed. The freezing of hydro-alcoholic liposomial suspensions did not alter the characteristics of liposomes according to microregion entrapment theory. Indeed, upon freezing the ethanol remained uniformly entrapped as a liquid in the solid microregions allowing liposomes to reorganize without undergoing to damages caused by ice crystals or cryo-concentration. Thus, the possible effect of protectants upon thawing was deepened on liposomes prepared by the thin film hydration method. Threalose and PVP at 0.5% m/v in mixture permitted to preserve liposome size distribution. According to DSC data, the protectant effect of threalose was attributed to formation of H-bonds with the polar heads of DPPC and in the case of PVP to the combination of H bonds formation and interaction with the lipid bilayer components. All freeze-dried products presented a moisture content lower than 1% and the ethanol content was negligible. The cake appearance was related to the ethanol content in the original nanosuspension. Up to 1%, the dried powder was “blown out” of the vial (“product ejection”) as a consequence of the poor cohesion within the cake. This defect was considered critical as it impacted on the product quality attribute. Indeed, the formation of too dispersed aggregates in the reconstituted sample prevented the DLS analysis. At residual ethanol content of 0.1%, lyophilized products had the same size and shape as the nanosuspension originally filled
Tipologia IRIS:
14 - Intervento a convegno non pubblicato
Elenco autori:
F. Selmin, S. Franzè, U. Musazzi, P. Minghetti, F. Cilurzo
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