2.2 Reverse Engineering the LTSL
2.2.4 Composition-Structure-Properties of Each Component
2.2.4.1 The Solid Phase Encapsulating Membrane
The encapsulating membrane provides the first two functions: to allow loading of a drug (e.g. doxorubicin), and to retain the drug in processing and upon i.v.
administration into the bloodstream. The properties that allow or relate to these functions are the Elastic Area Expansivity of the membrane (KA), its mechanical tensile strength (ts) and its internal dielectric constant (ofB2) that, together with the expansivity, limits the permeability of polar molecules and ions, but can let neutral doxorubicin through. (How this influences drug loading will be discussed in Section 2.2.4.3.)
Compositionally, the encapsulating membrane is composed of DiPal- mitoylPhosphatidylCholine (DPPC). Similar to SOPC, it contains a high proportion of carbons and hydrogens, in an empirical formula of C40H80NO8P (2 CH2s less than SOPC), which structurally form two C16 acyl chains. Both acyl chains are saturated, meaning they do not contain any C¼C double bonds.
These saturated chains can exert maximum van der Waals attraction on each Materials Science and Engineering of the Low Temperature Sensitive Liposome 43
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other in the solid phase, especially as C–C bond rotation is reduced at low relative temperatures giving a more all-transconformationalstructure.
Structurally, the solid bilayer is made up of crystalline grains with grain boundaries, as shown in Figure 2.5.
The grain structure of the liposome bilayer is evident from the faceted structure shown in Figure 2.5A, a Transmission Electron Microscope (TEM) image of the LTSL itself.65,66Also shown in Figure 2.5B are Scanning Electron Microscope (SEM) images of DSPC-PEG-Stearate monolayers on gas micro- particles,67and in Figure 2.5C is a fluorescent image of a 20-micron diameter gas microparticle monolayer where a fluorescent lipid (BODIPY FL PE) was included in the bilayers and segregated to the grain boundaries because it was not soluble-compatible with the solid grains of DSPC. It is interesting to see that the same micron-sized grains exhibited by the larger solid-lipid monolayers are still preserved as faceted structure even in the 100 nm diameter LTSL liposome, just scaled down with overall domain size.67
Property-wise, how strong might we expect this solid bilayer actually to be?
And how might we actually measure it? Using the micropipet technique, suction pressures in the milli atmosphere range can be applied to a single vesicle, and the membrane can be made to expand and eventually fail.55,68 In this experiment, shown in Figure 2.6A, a single GUV is aspirated by a micropipet, and a controlled suction pressure is applied to expand the single bilayer membrane into the pipet. As is also shown in Figure 2.6B, by measuring the length of the membrane projectionDL in the pipet, along with the external diameter of the vesicle (2Rv), and diameter of the pipet (2Rp) as a function of suction pressure DP, constitutive mechanical relations57 provide a direct measure of the membrane tension and the resulting area change,i.e. membrane tensile stresstand membrane area changeDA used to evaluate the stress/strain and the elastic area expansivity modulus KA.
Experiments like this have been carried out on many lipids and lipid mixtures.55,67–72As shown by Needham and Nunn68and reviewed by Kim and
A B C
Figure 2.5 Microstructure. Transmission Electron Microscope (TEM) image of a single 100 nm diameter LTSL containing doxorubicin (A); Scanning Electron Microscope (SEM) images of an 8 micron diameter gas micro- particle stabilized by a monolayer of DSPC : PEG-Stearate 9 : 1, and a higher magnification image of the grains and grain boundaries (B); and larger micro-gas-bubble monolayer (B20mM), where the lipid monolayer shells incorporate a fluorescent lipid that segregates to the grain boundaries, viewed under epifluorescence (C).
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Needham,69when taken to the limits of composition,i.e. 50 mol% cholesterol in a bilayer composed of a long (diC20) saturated chain phospholipid, membrane elastic moduli can reach several thousand mN/m and tensile strength can be 40 mN/m, which is equivalent to the compressibility and strength of bulk hydrocarbons and polyethylene.
Mechanically, the compressibility and the tensile strength have not been measured for DPPC, but we can show the effect of solidification of a bilayer on its mechanical properties by looking at the lower temperature transition of diC14 (dimyristoylphosphatidylcholine, DMPC) lipid that has been measured.73This lipid (Tm¼24.51C), its liquid and solid phases, including the area change at its pre- and main-transitions, was studied extensively by Needhamet al.73For this discussion, results showed that while the liquid La phase bilayer (at 291C,i.e. 4.51C above its main transition) had an elastic area dilation modulus (KA) of 145 mN/m, the solid Lbphase at 81C was much stiffer and stronger at 855 mN/m. Between 111C and 24.51C DMPC has a rippled Pb0 phase pre-transition region; namely, a low-enthalpy transition below the chain- melting transition linked to the formation of periodic ripples. Although ostensibly ‘‘solid’’, in this region the bilayer is still relatively soft (KA¼318 mN/m–228 mN/m measured at temperatures 201C and 14.51C, respectively).
For DPPC, the pre-transition region extends from 41.51C down to 35.51C.74 Thus, when extrapolated to DPPC, what these DMPC data show is that the DPPC bilayers as liposomes in the bloodstream at 371C are likely to be solid but still relatively soft, and only reach a relatively higher modulus, perhaps 1000 mN/m, at temperatures around, say, room temperature during processing.
Since membrane compressibility is directly related to its permeability,75 the nature of these mechanical states could be very important for determining the
A B
Figure 2.6 Vesicle Area Dilation Experiment. Giant Unilamellar Vesicle (GUV) (25 micron diameter) aspirated into the micropipet (8 micron diameter) ready for the expansion experiment, showing the measureable dimensions, external vesicle diameter Dv, pipet diameter Dpand membrane projection length in the pipet, Lp(the vesicle has a sucrose solution inside and a salt solution outside, creating a refractive index for better visualization) (A);
and constitutive equations for determining membrane tensiontfrom pipet suction pressure DP, and radii of vesicle and pipet, and the change in vesicle membrane area DA from the radii of pipet and vesicle, and the projection lengthDL (B).
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ability of the liposome to retain the drug, and indeed the H1ions that maintain its cationic nature. In the performance of the design then, we might expect compromised permeability in the bloodstream; later we will see if this is a concern. But, if an inherent leakiness might be the case, then why not make the membrane stronger? In other micromechanical experiments we have shown that the inclusion of cholesterol is the single biggest factor in increasing the elastic modulus of a lipid bilayer.68For example, KAof SOPC and SOPC : Cholesterol 1 : 1 isB200 mN/m and 1200 mN/m, respectively. For DPPC : Cholesterol 1 : 1 the modulus is 2500 mN/m, and so liposomes made from these membranes would be expected not only to retain drug better, but also remain in the bloodstream longer, because of their high resistance to expansion.76As shown in Figure 2.7, when the reticuloendothelial system is functional (i.e. not saturated with other lipids), liposomes circulate for longer, the higher their elastic modulus, indicating that less compressible interfaces resist opsonization.76 In fact, this strategy has been used to great effect for a vincristine liposome formulation. Sphingo- myelin : cholesterol 1 : 1 bilayers are very incompressible (KA¼1800 mN/m) and strong (ts¼23 mN/m),68,69 are extremely impermeable to water,75and actually circulate in the bloodstream for extended periods of time, forming the basis for a vincristine liposome that is required to release (leak) small amounts of drug very slowly during its circulation time.77 However, it has been shown by several techniques (deuterium NMR and Differential Scanning Calorimetry78and small- angle X-ray diffraction79) that the addition of cholesterol to DPPC bilayers totally abolishes the phase transition beyond 25 mol% cholesterol. Thus, by attempting to strengthen the membrane potentially to improve drug retention we defeat our object of creating a thermally responsive liposome.
Given the inherent encapsulation properties of lipid bilayers, if we are designing for thermal sensitivity and treatments for cancer, then the actual
Figure 2.7 Blood circulation half-life as a function of the membrane elastic modulus for conventional liposomes (non-PEGylated); data for unsaturated reti- culoendothelial system (RES).
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value of the transition temperature has to be the most important property. The choice of DPPC is governed by its transition temperature of 41.51C,74only a few degrees above body temperature (371C). Could other lipids suffice? Not as saturated PCs. The effect of2 CH2s per chain on the fully hydrated bilayer transition temperature can be seen by comparingTmof DPPC with that of the diC14 (24.51C) and diC18 (551C) lipids in the same homologous series,i.e. the next one down has a phase transition that is too low, and the next one up is much too high for hyperthermia treatment as the trigger. Mixing the lipids only serves to broaden the transition.74Introducing a double bond in one of the acyl chains can lower the transition temperature because it influences chain-chain packing. However, these effects are too large. For example, the DSPC tran- sition (551C) is reduced by 501C to 51C by just introducing one double bond in the 9 position of one of the C18 chains,i.e. as SOPC.
With further regard to lipid mixtures and their effect onTmand drug release, in 1978 Yatvinet al.38developed the first temperature-sensitive liposome based on the lipid DiPalmitoylPhosphatidylCholine (DPPC). As mentioned earlier, the reason was one ofproperty, since it has a transition temperature of 41.51C, just above body temperature (371C). However, Yatvinet al.’s formulation also contained the longer chain lipid DiStearoylPhosphatidylCholine, (DSPC) in a 7 : 1 DPPC : DSPC ratio. The addition of DSPC to the formulation raised the transition temperature of the ideal solid solution bilayer80 such that the liposome maximally released its encapsulated material in the temperature range of 43–451C39,40,46 Drug release rate was found to be slightly enhanced over non-transitioning bilayers,46but was still too slow for therapeutic use. Also the hyperthermic temperatures required were slightly higher than the clinically attainable range.81 As a consequence, further work and development of this thermal sensitive formulation all but ceased.81
In summary, while DPPC seems to be an ideal host lipid with a transition temperature just above body temperature, where the lipid bilayer can become transiently more permeable, its enhanced permeability at the phase transition is not much above a liquid phase lipid at the same temperature.46Attempts to increase drug retention by making the bilayer stronger and less compressible, by, say, mixing in cholesterol, only serves to abolish the transition altogether, and including a longer acyl chain lipid like DSPC raises the Tm beyond the attainable mild hyperthermia limit (ofB421C) and slightly broadens it, again producing undesirable properties. The answer to this materials choice dilemma was to include a permeabilizing component,i.e. a non-bilayer lysolipid.17