Study on the in situ aggregation of liposomes with negatively charged phospholipids for use as injectable depot formulation

Abstract

Compared to conventional parenteral formulations injectable depot formulations, owing to a sustained drug release, offer several advantages, such as a reduced dosing frequency - and consequent improved compliance - or a predictable release profile. Additionally, fluctuations in the drug blood level may be smoothened and consequently side effects reduced. Because of their capability to encapsulate water soluble drugs and their very low toxicity profile liposomes comprising phospholipids, most certainly represent a vehicle of choice for subcutaneous (s.c.) or intramuscular (i.m.) administration typical for depot injections too. In the past, especially liposomes containing negatively charged phosphatidylserines were investigated regarding their aggregation and fusion behavior upon addition of calcium ions. Liposomes need to have a large size to prevent fast removal from the s.c. or i.m. injection site to make them useful as depot vehicle. In order to obtain such large liposomes, aggregation of smaller liposomes may be considered. Aim of the present study was to induce and investigate controlled aggregation of vesicles containing other negatively charged phospholipids besides phosphatidylserines upon mixing with a solution of divalent cations. L-α-phosphatidylcholine from egg (EPC) liposomes formulated with 25 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) or 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG) proved to be possible lipid-based depot candidates due to their strong aggregation induced by calcium and magnesium cations. Different aggregation profiles with both cations could be observed. Morphological investigations of the aggregates showed that individual liposomes remain stable in the aggregates and no fusion occurred. A fluorescence-based fusion assay confirmed these results. Differential scanning calorimetry measurements supported the findings of the diverse aggregation profiles with calcium or magnesium owing to different binding sites of the cations to the lipid molecules.

Keywords: Aggregation; Depot formulation; Liposome; Negatively charged phospholipid.