2.4 Performance-in-Service
2.4.1 Performance-in-Service: in vitro
2.4.1.4 Drug Release Mechanism
In understanding the drug release mechanism it is important to realize that each of the components influences the material characteristics of mechanical integrity and strength, interfacial steric stability, thermal melting and membrane grain- and nanostructures. In turn, they are expected to control and allow the manipulation of many of the desired pharmaceutic and clinical- performance criteria of the liposomes, such as the rate of drug loading, circu- lation half-life, drug retention and the temperature and rate of thermally triggered drug release.
As shown above and in the several publications and reviews that have chronicled its development and testing,15,16,94–97compared to DPPC alone, the MSPC-based formulation releases drug within seconds of being heated to its main acyl melting phase transition, because each of these acyl-chain-compatible components are stably (perhaps ideally) mixed in the gel phase of a lipid bilayer and then create what appear to be membrane nanopores at the phase transition, probably at grain boundaries in the melting lipid.98It appears that the presence of both MSPC and DSPE-PEG are important to the ultrafast permeability of the LTSL formulation. Both Mills and Wright43,49 had shown that without lysolipid, even with DSPE-PEG present, doxorubicin permeability was low.
In the absence of MSPC, a DPPC : DSPE-PEG2000(increasing DSPE-PEG2000 from 2 mol% to 20 mol%) formulation only releasedo20% doxorubicin at the lipid phase transition, which was probably due to membrane bound doxo- rubicin (Figure 2.16A). For comparison, the release data for Dox-loaded standard LTSL formulation are also presented in Figure 2.16A (open circles).
Then, in another experiment, where the membrane composition was DPPC : MSPC (90 : 10),i.e. with 0% DSPE-PEG2000, significantly slower drug
A B
Figure 2.16 The unexpected role of DSPE-PEG2000. Percent Dox released into 20 mM HBS at Tm (41.31C) as a function of DSPE-PEG2000 in the absence of MSPC (A), and effect of inclusion of DSPE-PEG2000on % Dox release at 41.31C from LTSL with increasing membrane mol percents of DSPE-PEG2000in the presence of MSPC (B).
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release than the LTSL formulation was observed (Figure 2.16B). It is only with the inclusion of just 1.3 mol% DSPEG-PEG2000that the rate of drug release is increased at the transition temperature.
Even though the DSPE-PEG2000lipid has two acyl chains (Figure 2.3), it has a shape factor similar to MSPC in that the head-group is much larger than the tail-group due to the polyethylene glycol polymer. Thus, DSPE-PEG forms micelles in aqueous solution having a cmc that is actually similar to MSPC of around 1ml.99 It could, in principle, support positive curvature in lipid membranes. We therefore hypothesized that the molecular shape of DSPE- PEG2000 might make it an important part of lysolipid pore stabilization, and therefore help control, to some extent, the triggered-drug release from LTSL.
Thus, Dox release from DPPC : MSPC liposomes with 0 mol% DSPE- PEG2000was significantly slower than from the LTSL formulation, suggesting that PEG-lipid could be an important factor in stabilizing the postulated permeabilizing pores. Indeed, incorporating only 1.3 mol% of the PEG-lipid increased the release rate and amount to values similar to the LTSL formu- lation. So here was a situation where a component, DSPE-PEG2000, which was originally included in order to enhance circulation time in the bloodstream, was now providing a second and very important function of enhancing the permeability of the lysolipid-containing bilayers. However, it does not appear to enhance the phase transition permeability of DPPC or form putative nanopores if it is the only included component.
As previously discussed in detail42 and summarized in Figure 2.17, we therefore propose the following mechanism for drug retention, its possible leakage during the blood-borne transport phase and its ultrafast release at the solid-liquid transition of the LTSL membrane.
Drug Retention and Possible Leakage at 37 1 C
Figure 2.17A shows the mixed-lipid bilayer in its solid phase at 371C. As modeled by Mouritsenet al.36,100the chain mismatches between solid, mostly translipids do not line up exactly with the more liquid-like chains of the grain boundary region. There is a pH gradient across bilayer, and doxorubicin is still in the protonated-unprotonated equilibrium at pH 5.5 (pH is known to rise slightly from its initial value of 4.0 due to Dox loading). Consequently we might expect good retention of the drug and a low doxorubicin permeability through the bilayer matrix itself, and even through the grain boundary defects.
However, as discussed earlier, DPPC has a pre-transition around 351C, at which point the bilayer, although still solid, enters a slightly less compressible phase (Pb0). Thus, while the unprotonated form of doxorubicin itself is in low concentration and doxorubicin is actually in crystal form, its retention in the liposome bothin vitro and in vivocould hinge critically on the ability of the bilayer to retain hydrogen ions. Perhaps more significantly, then, any H1 permeability through defects or solid phase bilayer (blue arrows) would deplete the hydrogen ion concentration inside the liposome, drive the equilibrium towards more unprotonated doxorubicin and the now membrane-soluble
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doxorubicin could follow. This is what we think happens in the bloodstream, where doxorubicin has been shown to leak out of the liposome over a period of a few hours. The presence of some bilayer soluble doxorubicin (red ‘‘Dox’’) in the bilayer would also account for the low-level release of doxorubicin in the in vitroassays for drug leakage (Figures 2.15 and 2.16).
Rapid Release at 41.5 1 C – Role of Nanopores
With MSPC and DSPE-PEG2000in the bilayer, as depicted in Figure 2.17B, the bilayer in the phase transition region acquires enhanced permeability through a purported MSPC pore. As the transition temperature is approached and the grain boundaries begin to melt, lateral lipid transport could well be increased and could allow more lysolipid to assume its preferred curvature (i.e. as a convex micelle), relaxing the planar bilayer structure by forming lysolipid-lined nanopores. With a few mol% of DSPE-PEG2000in the bilayer, this MSPC pore
B A
Figure 2.17 Proposed mechanism for drug retention, leakage and thermally triggered (smart) release. Retention and possible leakage at 371C (A) and release at 411C (B).
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is stabilized by PEG-lipid. As a consequence, the hydrogen ion gradient rapidly equalizes, DoxH1 comes out in seconds (large red arrow), as does any remaining embedded Dox in the bilayer. From dextran permeability measurements and other calculations,43the size of these nanopores appears to be B10 nm in diameter, more than large enough to allow the very rapid transport seen for CF, dithionite and doxorubicin.