Haemocompatibility improvement of metallic surfaces by covalent immobilization of heparin-liposomes.

Stainless steel surfaces were processed by means of plasma enhanced chemical vapor deposition (PE-CVD) fed with acrylic acid vapors in order to functionalize them with carboxyl groups, which were subsequently activated for covalent immobilization of heparin-loaded (HEP) NH(2) group-functionalized (Fun) nanoliposomes (NLs). Empty Fun or HEP non-functionalized (control) NLs were used as controls. NLs were characterized for mean diameter, surface charge and heparin encapsulation/release. Different lipid compositions were used for NL construction; PC/Chol (2:1mol/mol) or PC/Chol (4:1mol/mol) (fluid type vesicles) [which allow gradual release of heparin] and DSPC/Chol (2:1mol/mol) (rigid type vesicles). Surface haemocompatibility was tested by measuring blood clotting time. Platelet adhesion on surfaces was evaluated morphologically by SEM and CLSM. The haemocompatibility of plasma-processed surfaces was improved (compared to untreated surfaces); Fun-HEP NL-coated surfaces demonstrated highest coagulation times. For short surface/blood incubation periods, surfaces coated with Fun-HEP NLs consisting of PC/Chol (2:1) had higher coagulation times (compared to DSPC/Chol NLs) due to faster release of heparin. Heparin release rate from the various NL types and surface platelet adhesion results were in agreement with the corresponding blood coagulation times. Concluding, covalent immobilization of drug entrapping NLs on plasma processed surfaces is a potential method for preparation of controlled-rate drug-eluting metallic stents or devices.

Haemolytic activity of liposomes: effect of vesicle size, lipid concentration and polyethylene glycol-lipid or arsonolipid incorporation.

The haemolysis caused by various types of liposomes was measured after incubation of liposomes with human red blood cell (erythrocyte) suspension. Liposomes composed of phospholipids and containing or not arsonolipids (arsonoliposomes) were tested. In some cases liposomes that were coated with polyethylene glycol (MW 2000), which were formulated by including 8 mol% DSPE-PEG2000 in their lipid membrane, were used. Multilamellar vesicles were prepared by the thin film hydration technique (conventional liposomes) or by the one-step technique (arsonoliposomes). Sonicated vesicles were produced by probe sonication of the initial liposome preparations. Phospholipid concentration in the liposome dispersions were measured by the Stewart assay, and adjusted accordingly. Haemolysis was measured after incubating 100 microl of liposome dispersions with 900 microl of red blood cell suspension (blood) for 1 h. The results reveal that the haemolysis caused, when liposomes are incubated in blood at concentrations below 0.16 mg (lipid)/ml (blood), was minimum. Only in case of Pegylated arsonoliposomes, significant haemolysis percents were observed. At higher lipid concentrations, 0.38 or 0.6 mg/ml, the haemolysis caused by arsonoliposomes was substantially increased, even in the cases of non-Pegylated arsonoliposomes. In most cases, especially when arsonolipid-containing liposomes were evaluated, vesicle size also had considerable effect on vesicle-induced haemolysis. Nevertheless, at concentrations which are relevant with liposomal drug administration in humans, all formulations tested demonstrated negligible haemolysis.