The preparation of tissue-type Plasminogen Activator (t-PA) containing liposomes: entrapment efficiency and ultracentrifugation damage.
In this study, a method was developed for the efficient entrapment of active tissue-type Plasminogen Activator (t-PA) into liposomes. Experimental conditions were varied to optimize t-PA entrapment: different buffer solutions were used (pH 4 and 7.5), the effect of the incubation concentrations of phospholipid (PL) and t-PA was monitored and the influence of liposome-size was examined. Furthermore, the effect of ultracentrifugation on t-PA containing liposomes was determined in the presence and absence of Tween 80. t-PA entrapment strongly depended on experimental conditions and ranged from 30 up to 90%. Almost quantitative+ (90%) entrapment (entrapment percentage defined as absolute entrapment (IU t-PA/mumol PL) divided by total incubation ratio (IU t-PA/mumol PL), times 100%) was obtained in Hepes buffer pH 7.5, devoid of arginine, with low ionic strength. Ultracentrifugation, used for removal of non-entrapped t-PA, was shown to have a damaging effect on the liposomes (especially in the presence of 0.05% Tween 80), leading to t-PA loss. However, because acceptable alternatives were not available, ultracentrifugation was used during this study. Therefore, the encapsulation-percentage values shown in this study are in fact underestimates for the true entrapment of t-PA.


CONCLUSIONS
almost quantitative t-PA entrapment in liposomes can be achieved by selecting the proper milieu and inducing a strong interaction between t-PA and bilayer.
Heeremans
J L
JL
Department of Pharmaceutics, Utrecht University, Netherlands.
Gerritsen
H R
HR
Meusen
S P
SP
Mijnheer
F W
FW
Gangaram Panday
R S
RS
Prevost
R
R
Kluft
C
C
Crommelin
D J
DJ
D002021:Buffers; D004339:Drug Compounding; D004355:Drug Stability; D005615:Freezing; D006801:Humans; D008081:Liposomes; D010316:Particle Size; D010743:Phospholipids / Q000737:chemistry; D011136:Polysorbates; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000008:administration & dosage* / Q000737:chemistry; D014461:Ultracentrifugation
D002021:Buffers; D008081:Liposomes; D010743:Phospholipids; D011136:Polysorbates; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator