Search

The influence of the water-soluble peptide, desmopressin (DDAVP) and its dipalmitoylated prodrug derivative (DPP) on the thermal behaviour of three different saturated phosphatidylcholine lipid membranes was investigated by differential scanning calorimetry. For lipid membranes composed of dimyristoyl, dipalmitoyl and distearoyl phosphatidylcholines the addition of DDAVP at concentrations of up to 10 mol% resulted in an insignificant change in the thermodynamic phase behaviour. In contrast, the dipalmitoylated DPP prodrug caused major changes on the lipid membrane phase behaviour manifested as a drastic decrease in the heat capacity peak height and a concomitant broadening of the main phase transition as well as a decrease in the transition enthalpy. In addition, the main phase transition temperature was slightly decreased and the pre-transition of the three phosphatidylcholines was abolished when DPP was present.

Polymer grafted PEG-liposomes have come into use as drug-delivery systems with improved therapeutic profiles. However, very little is known about the morphological instability of PEG-liposomes due to enzymatic degradation. To gain further insight into the effect of PEG lipopolymer-concentration on the catalytic activity of a liposome-degrading enzyme, phospholipase A2 (PLA2)-catalyzed phospholipid hydrolysis of PEG-liposomes has been investigated. The temperature dependence of the PLA2 lag-time, denoting the time required before a sudden increase in enzymatic activity takes place, has been determined for submicellar amounts of dipalmitoylphosphatidylethanolaminyl-poly-(ethylene glycol) (DPPE-PEG2000) incorporated into unilamellar dipalmitoylphosphatidylcholine (DPPC)-liposomes. The measurements demonstrate a significant reduction in the lag-time over broad temperature ranges. The results suggest that a close relationship exists between PLA2 catalyzed lipid hydrolysis and lipid-membrane composition, which moreover is of major importance for the overall morphological stability and the release of encapsulated material from the polymer-grafted PEG-liposomes.

It is well known, that a prolonged liposome circulation time can be achieved by incorporation of lipopolymers into the lipid membrane thereby reducing interactions with destabilizing factors in the blood stream, e.g. phagocytic cells and lipoproteins. However, very little is known about the enzymatic degradation of steric hindered liposomes introduced into body fluids. In this study, the blood clearance and the PLA2 catalyzed degradation of unilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes incorporated with increasing amounts of dipalmitoylphosphatidylethanolamine-polyethyleneglycol (DPPE-PEG), was investigated. The results demonstrated an increase in PLA2 activity for increasing amounts of lipopolymer in the lipid membrane, while the liposome blood clearance was prolonged by incorporation of DPPE-PEG into the liposomes. Hence, these results suggest that it may be possible for long circulating liposomes to obtain a site specific liposome degradation and release of drug substance in tissue with high levels of PLA2.

The effect of poly(ethylene glycol)-phospholipid (PE-PEG) lipopolymers on phospholipase A(2) (PLA(2)) hydrolysis of liposomes composed of stearoyl-oleoylphosphatidylcholine (SOPC) was investigated. The PLA(2) lag-time, which is inversely related to the enzymatic activity, was determined by fluorescence, and the zeta-potentials of the liposomes were measured as a function of PE-PEG lipopolymer concentration. A significant decrease in the lag-time, and hence an increase in enzymatic activity, was observed with increasing amounts of the negatively charged PE-PEG lipopolymers incorporated into the SOPC liposomes. The enhancement of the PLA(2) enzymatic activity might involve a stronger PLA(2) binding affinity towards the negatively charged and polymer covered PEG liposomes.

Due to an increase in the activity of phospholipase A(2) (PLA(2)) in various inflammatory diseases, this enzyme may play a key role in the degradation of liposomes and the subsequent release of drug when PEG-liposomes passively target inflammatory tissue. The activity of mammalian secreted phospholipase A(2) (sPLA(2)) in casein stimulated peritoneal fluid was tested toward liposomes of different compositions. Early results indicate only a slight degradation of conventional dipalmitoylphosphatidylcholine (DPPC) liposomes as well as DPPC liposomes incorporated with different concentrations of PEG(2000). However, the DPPC degradation increased to 7% when inclusion of 30 mol% phosphatidylethanolamine (PE) in the lipid bilayer. The increase in degradation may be due to an improvement of the substrate - as it is well known, that PE is a better substrate for the mammalian sPLA(2) than PC. Incorporation of PE into the bilayer may increase the binding properties of the bilayer resulting in improved conditions for the enzymatic attack by sPLA(2). In addition, inhibitory zones of Staphylococcus aureus in an agar diffusion test showed that PLA(2) from Crotalus atrox venom was able to catalyze the release of gentamicin from PEG-liposomes. In conclusion, this study suggest that degradation of the lipid bilayer of PEG-liposomes by PLA(2) result in release of incapsulated drug, e.g. gentamicin and inclusion of PE in the liposomal bilayer, may enhance the activity of the mammalian sPLA(2) toward liposomes composed of DPPC.

The interaction between a small positively charged peptide with a N-terminally linked acyl chain and dipalmitoylphosphatidylcholine–dipalmitoylphosphatidylserine (DPPC–DPPS) lipid membranes has been studied by means of fluorescence resonance energy transfer. Two different lipid compositions were used: a neutral membrane (100 mol% DPPC), and a negatively charged membrane (30 mol% DPPS in DPPC). The fluorescence resonance energy transfer results reveal that the peptide associates with both types of membranes. Furthermore, it is found that the slope of the titration curve for the negatively charged membranes is much steeper than that for the neutral membranes. This indicates a higher binding affinity of the acylated peptide towards negatively charged lipid membranes as compared with neutral lipid membranes.

This study investigates the screening effect of poly(ethylene glycol)-phospholipids (PE-PEG) on the interaction of avidin with PEGylated liposomes containing surface-bound biotin ligands. The influence of grafting density and lipopolymer chain length is examined. A simple fluorescence assay involving a receptor-mediated fluorescence increase of BODIPY-labeled avidin upon binding to biotinylated lipids is employed to study the screening effect of submicellar concentrations of 1,2-dipalmitoyl- sn -glycero-3-phosphatidylethanolamine- N -[poly(ethylene glycol)-2000] (PE-PEG 2000 ) and 1,2-dipalmitoyl- sn -glycero-3-phosphatidylethanolamine- N -[poly(ethylene glycol)-5000] (PE-PEG 5000 ) incorporated into 1,2-dipalmitoyl- sn -glycero-3-phosphatidylcholine (DPPC) liposomes. The results show that incorporation of lipopolymers into DPPC lipid bilayers reduces binding of avidin to the biotinylated liposomes, and it is found that the screening effect of PE-PEG 5000 is stronger than that for PE-PEG 2000 . Thus, the results reveal that both the grafting density and the polymer length of the PE-PEG lipopolymers are of importance for the ability of water-soluble macromolecules to reach the surface of PEG liposomes. Furthermore, it is found that none of the lipopolymers completely prevents avidin from reaching the surface-bound biotin ligands.