Gry Stensrud

Liposomes as carriers of amphiphilic gadolinium chelates: the effect of membrane composition on incorporation efficacy and in vitro relaxivity

The effects of membrane composition (phospholipid type and amount of cholesterol), liposome size, drug/lipid ratio (loading) and nature of the amphiphilic gadolinium (Gd) chelate on the incorporation efficacy and magnetic resonance (MR) contrast efficacy (longitudinal (T1) relaxivity) were investigated using a fractional factorial design. A highly lipophilic Gd-chelate was required to ensure complete liposome incorporation. High T1-relaxivity was obtained by using liposomes composed of cholesterol and phospholipids with short acyl chain lengths (dimyristoyl phosphatidyl choline (DMPC) and dimyristoyl phosphatidyl glycerol (DMPG). Two key factors, the loading of Gd-chelate and the amount of cholesterol in small-sized DMPC/DMPG liposomes, were studied further in a central composite optimising design. A robust high relaxivity region was identified, comprising high loading of cholesterol and Gd-chelate. However, the highest T1-relaxivity (52 mM(-1) s(-1)) was found in an area containing no cholesterol and low content of Gd-chelate. Nuclear magnetic resonance dispersion (NMRD) profiles were obtained for five of the liposome compositions from the optimising design, and high relaxivity peaks in the 20 MHz region confirmed the presence of Gd-chelates with a long tau(R). A liposome formulation was selected for surface modification with polyethylene glycol (PEG), without having any effect on the T1-relaxivity.

Formulation and characterisation of primaquine loaded liposomes prepared by a pH gradient using experimental design

The effect of different formulation factors (lipid type, cholesterol, charge, internal buffer capacity, drug-to-lipid incubation ratio) on the encapsulation efficiency and size of primaquine liposomes (SUVs) in response to a pH gradient was investigated by a fractional factorial screen ing design. Three of the factors (charge, internal buffer capacity, drug -to-lipid incubation ratio) were further studied in a Box--Behnken optimisation design. The lipid type was the most important parameter followed by the drug-to-lipid incubation ratio, buffer capacity, cholesterol and charge. Several of the interactions wer e important. In the optimisation design a robust region with high encapsulation efficiency (>95%) was obtained for DSPC: 33.33 mol% cholesterol-liposomes at high internal citrate concentration (200 mM) by maintaining the drug-to-lipid incubation ratio below 0.15:1 (mol:mol) and varying the charge incorporation between 2 and 10%. In order to achieve long-term stability and sterility, the liposomes were lyophilised followed by gamma irradiation. The pH gradient was maintained during this treatment with little chemical degradation of the substances. The final preparation consisted of three separate vials with lyophilised liposomes, solid state primaquine and hydration medium.

Novel high relaxivity colloidal particles based on the specific phase organisation of amphiphilic gadolinium chelates with cholesterol.

To obtain high T(1)-relaxivity colloidal particles with a simultaneously high loading of amphiphilic Gd-chelates, a novel drug dosage form based on the phase organisation of amphiphilic gadolinium chelates with cholesterol was developed. In order to find a formulation, which exhibit both high T(1)-relaxivity and gives small particles a D-optimal mixture design (experimental design) was applied. Gadolinium 1,4,7-tris(carboxymethyl)-10-(2-hydroxyhexadecyl)-1,4,7,10-tetraazacyclododecane (Gd-HHD-DO3A) and cholesterol at approximately equimolar ratio proved to form thermodynamic stable disc-like colloidal particles as seen by cryo-electron micrographs. T(1)-relaxivity of these particles was typically around 20mM(-1)s(-1) and the size below 100 nm (photon correlation spectroscopy (PCS)). The particles do most probably not interact with blood components as no change in T(1)-relaxivity was observed when the particles were mixed with whole blood. The particles were stable at room temperature for at least 6 months.

Toxicity of gamma irradiated liposomes. 1. In vitro interactions with blood components

Gamma irradiation is a potential technique for sterilisation of liposome suspensions. Unfortunately, gamma irradiation may result in chemical degradation of the phospholipids and the toxicological aspects have to be considered. The effects of liposome composition and gamma irradiation on the interactions of the liposomes with the hemostatic mechanisms (hemolysis, aggregation and coagulation) were studied. Non-irradiated liposome suspensions showed no hemolysis of erythrocytes. After irradiation, up to 3.1% hemolysis was measured. Least hemolysis was observed with irradiated liposomes composed of unsaturated or charged phospholipids. The negatively charged DSPG-liposomes (both non-irradiated and irradiated) induced aggregation of platelets as observed by the spectrophotometric method. However, no aggregates were seen in the microscope or measured by the aggregometer. Negatively charged liposomes also affected the coagulation cascade where prolonged coagulation times were measured. Irradiation of the liposome suspensions resulted in even longer coagulation times. The prolonged coagulation times correlated to some extent with the measured binding and depletion of calcium from plasma by the negatively charged liposomes.

Toxicity of gamma irradiated liposomes.: 2. In vitro effects on cells in culture

In this study, the effects of liposome composition and gamma irradiation on their interactions with cell cultures were studied. The cytotoxicity test and the growth inhibition test clearly revealed toxic effects of liposomes composed of unsaturated phospholipids and gamma irradiation of these preparations enhanced their toxic effects. The murine fibroblast cell-line L 929 was less affected compared to the macrophage cell-line RAW 264 with a higher endocytic capacity. On the other hand, both gamma irradiated and non-irradiated liposomes composed of saturated phospholipids were non-toxic for the cells and irradiation did not affect their drug delivery properties. Hence, it seems that gamma irradiation is appropriate for sterilisation of these liposomes.

Effects of Gamma Irradiation on the Physical Stability of Liposomal Phospholipids

AbstractThe effects of liposome composition and gamma irradiation on the phase transition, size, zeta potential and pH were investigated using factorial designs. In addition, the effect of irradiation on the leak-in rate of calcein was evaluated for one of the liposome composition. The liposomes were stored for 6 months in order to reveal any possible long term effects. The phospholipids used were dipalmitoyl phosphatidyl choline (DPPC) or egg phosphatidyl choline (egg PC). Charge was introduced to the liposomal bilayers by the addition of 10% dipalmitoyl phosphatidyl glycerol (DPPG) or egg phosphatidyl glycerol (egg PG). The liposome-suspensions were obtained by the extrusion method. After gamma irradiation changes in the phase transition, zeta potential and pH of the liposomes were observed. The size of the liposomes was not affected by the irradiation, but the irradiation prevented the neutral DPPC-liposomes from aggregation. This was confirmed by cryo-electron microscopy. No change in the leak-in rate...

Erratum to: “Effects of Gamma Irradiation on the Physical Stability of Liposomal Phospholipids” [J. Lipos. Res. 7, (1997) 503-528]

AbstractThe effects of liposome composition and gamma irradiation on the phase transition, size, zeta potential and pH were investigated using factorial designs. In addition, the effect of irradiation on the leak-in rate of calcein was evaluated for one of the liposome composition. The liposomes were stored for 6 months in order to reveal any possible long term effects. The phospholipids used were dipalmitoyl phosphatidyl choline (DPPC) or egg phosphatidyl choline (egg PC). Charge was introduced to the liposomal bilayers by the addition of 10% dipalmitoyl phosphatidyl glycerol (DPPG) or egg phosphatidyl glycerol (egg PG). The liposome-suspensions were obtained by the extrusion method. After gamma irradiation changes in the phase transition, zeta potential and pH of the liposomes were observed. The size of the liposomes was not affected by the irradiation, but the irradiation prevented the neutral DPPC-liposomes from aggregation. This was confirmed by cryo-electron microscopy. No change in the leak-in rate...