A lysosome-rich fraction from rat liver normally exhibits latency with respect to enzyme activity in the range 10-20% of the total activity in that fraction (Goldman and Kaplan, 1973). However when saline controls were incubated with the light mitochondrial fraction, NAGase release was variable and in some cases > 20% was detected (figures 5.3a-c). These variable levels may have resulted from lysosome instability after their addition to 250mM sucrose at different pH values and due to premature lysosomal breakage incurred during the fractionation procedure.
NAGase release caused by PEI (0.5mg/mL) at pH values o f 5.0 and 6.5 (when incubated on the outside of lysosomal membranes) showed substantial NAGase release (> 80%) after 30min and Ih. In contrast NAGase release caused by ISA 22 (0.5mg/ml) (figure 5.3b) and ISA 1 (0-2mg/mL) (figure 5.4) (when incubated on the outside of lysosomal membranes) at pH values 5.0-7.4 was not significantly different to the controls. The ability of PEI to cause lysosomal membrane breakage in a pH-dependent manner was surprising since experiments performed by Klemm et al, (1998) (described in the introduction to this chapter) demonstrated that although PEI caused lysosomal membrane perturbation, this did not occur in a pH-dependent manner. Although the concentration of PEI used by Klemm (90pg/mL) was much lower than the concentration of PEI used in this study (0.5mg/mL), a correlation between both sets of results was still expected. The protonation level o f PEI increases from 20-45% between pH values of 7 and 5 (Remy et al, 1998). Therefore the increased membrane breakage at pH values o f 5 and 6 could be due to the increased cationic nature of PEI. A second possibility for the increased free activity of NAGase when incubated with PEI at low pH values is due to the increased activity of NAGase at low pH values. As this enzyme is lysosomal, its pH optimum has been reported to be in the pH range 4.5-6.0 (Barrett and Heath, 1977).
C h apter 5. D irect eviden ce f o r P A A -in du ced lysosom al m em brane rupture
%
y
% : ■ ^ c 1'q
j
' - W \ - { < ■■ yFigure 5.7 Morphology of lysosomes after incubation in the presence of the free radical generating system (xanathine-xanathine oxidase) (from Dechameux et al, 1992).
C h a p te r 5. D ir e c t e v id e n c e f o r P A A -in d u c e d ly s o s o m a l m e m b ra n e ru p tu r e
No correlation was found between the RBC lysis model for membrane disruption and the lysosomal membrane disruption model (polymer on outside) demonstrated in this study. When PEI (1 mg/mL) was incubated with isolated RBCs at pH values of 5.5, 6.5 and 7.4, lysis of RBCs was not pH-dependent (Wan et al, 2002). However ISA 23, which was previously shown to lyse RBCs at pH 5.5 and 6.5 but not at pH 7.4 (Richardson et al, 1999a), was unable to cause NAGase release in a pH- dependent manner. A specific reason for this was not determined, however, as lysosomal membranes have a very different morphology to RBCs (discussed in the introduction to this chapter), polymer interaction with the outer lysosomal membrane could be very different to their interaction with a RBC membrane.
This model describes polymer interaction with the outer lysosomal membrane, which is effectively the wrong face of the membrane. The physiological model assesses the effect o f incubating polymers with the inner lysosomal bilayer, after their endocytic uptake and subsequent trafficking to lysosomes. As NAGase release was shown quantitatively to occur using this method (figure 5.5), it is possible that PAAs preferentially interact with the inner lysosomal bilayer compared to the cytoplasmic- facing lysosomal membrane. Lysosomal membrane perturbation may also have been observed with this model due to a higher concentration of ISA 1 inside the lysosome than the model describing polymer interaction with the outer lysosomal membrane. Endocytosis of ISA 1 causes its continuous delivery to the lysosomal compartment, thus it will be increasingly concentrated on this internal surface. This theory has also been proposed by Klemm et al, (1998) to explain the differences in PEI concentration required to cause efficient lysosomal membrane destabilisation and gene transfection. Transfection efficiency was reported to be maximal at PEI concentrations o f 2.5pg/mL. In these transfection experiments PEI was endocytosed, and therefore would be concentrated in intracellular compartments. However, when PEI was incubated with the outer lysosomal membrane, PEI concentrations ~ 20 times greater were required to cause significant NAGase release (Klemm et al, 1998).
A marked change in the morphology of lysosomes containing ISA 1 compared to control lysosomes was observed from TEM studies (figure 5.6). This also suggested that the inner lysosomal bilayer was compromised by ISA 1 to allow release of the densely staining lysosomal contents out o f the lysosome. Further evidence for the ability o f PAAs to disrupt lysosomes after their endocytic uptake was demonstrated when lOmg/kg '^^I-labelled ISA 4 was administered i.v. to rats. At different times post injection differential subcellular fractionation was performed on the removed liver.
C h a p te r 5. D ir e c t e v id e n c e f o r P A A -in d u c e d ly s o s o m a l m e m b ra n e ru p tu re
60min post-injection, ~ 20% radioactivity was found in the cytosolic fraction, compared to < 10% 30min post-injection. The appearance o f the polymer in the cytosolic fraction was also accompanied by an increase in NAGase in this fraction (~ 15%). These experiments suggested that endocytosed ISA 4 weis able to destabilise lysosomal membranes leading to release of ISA 4 and NAGase into the surrounding medium (Richardson, 1999c; Pattrick et al, 2002).
After endocytic uptake of ISA 1 (25mg/kg), vesicles were observed by TEM without densely staining lumen. This again suggested their membranes were destabilised by ISA 1.
It is essential that any non-viral vector developed for i.v. administration should not cause toxicity to normal cell membranes or cause breakage o f the lysosomal membrane following endocytic uptake. Excess lysosomal damage leads to cell death due to the degradative action of lysosomal enzymes released into the cell cytosol (Ichihara et al, 1991). Indeed, substantial polymer-mediated cytotoxicity was observed when PEI (Mw 25,000) was incubated in vitro with 293T cells after 2h (IC50 7pg/mL)
(Lee et al, 2001). PAAs, however, are relatively non-toxic to cells in vitro (IC50 >
2mg/mL, Ranucci et al, 1991), and doses up to lOOmg/kg ISA 4 administered in vivo
have shown no toxicity. Therefore, taken together with the indirect evidence for PAA- mediated endosomolytic ability discussed in chapters 3 and 4, it is likely that PAAs are mediating both endosomal- and lysosomal- membrane perturbation.
5.5 Conclusions
Although the PAAs ISA 1 and 23 did not to cause lysosomal rupture when incubated on the cytoplasmic face of the lysosomal membrane, these experiments indicated PAA-mediated lysosomal membrane perturbation after internalisation into rat liver lysosomes following administration of doses > lOmg/kg. The mechanism(s) responsible for polymer-mediated lysosomal membrane and endosomal membrane destabilisation must be characterised. The endosomal and lysosomal membranes have common origins although they are different in composition. Therefore to continue these studies further it is necessary to isolate endosomes and determine how PAAs interact with them.
In order to move the development of PAA-toxin conjugates into clinical development, it was important to further investigate the intracellular trafficking of PAAs alone and in combination with the plant toxin gelonin. In the next chapter the
C h a p te r 5. D ir e c t e v id e n c e f o r P A A -in d u c e d ly s o s o m a l m e m b ra n e ru p tu r e
synthesis and visualisation o f fluorescent-labelled PAAs and gelonin after endocytic uptake is described.
C h a p te r 6. E n d o c y tic u p ta k e o f l a b e lle d P A A s a n d g e lo n in