Resultados y análisis de la pendiente del terreno

The amphiphilic property of lipids allows them to undergo self-assembly to form complex aggregates in response to the hydrophobic effect/^ The formation of these aggregates is a concentration dependent phenomenon, and occurs when an amphiphile exceeds a certain concentration referred to as the critical micelle concentration (CMC).“

The variety o f aggregates that lipids can adopt is exemplified by phospholipids, which have been extensively studied for decades/^'^^ Phosphatidylethanolamine (PE) lipids form a wide variety o f complex higher order aggregates in aqueous solutions.^®’^^’^^ PE lipids are non-lamellar (non-bilayer) phase forming lipids and hence their role in gene transfer is of intense interest.^^'^^ These lipids have a wide variety of

associated lipid phase structures (Figure 1.2.15) and these are classified as follows

L, One dimensional lamellar; H, Two dimensional hexagonal; P, Two dimensional oblique; Q, Three dimensional cubic; C, Three dimensional crystalline.

Furthermore the hydrocarbon chains can be described as a (disordered or fluid) and p (untilted ordered or gel).

A single PE lipid is unlikely to exhibit all of these phases, however Seddon et a l

demonstrated that the lipid didodecylphosphatidylethanolamine, coexisted in crystalline

and fluid phases in temperatures of up to 83 °C.^ Furthermore, Seddon and co­

workers observed that reductions in pH or increases in ionic strength induces transitions between phases at lower temperatures.^^ Transitions between the phases can also be induced by changes in lipid concentration and temperature.^'

1 Introduction Gene Therapy

B ilay er L„

I n te r b ila v e r C o n ta c t

I

>oo<

In v e rte d c u b ic P h ase

\

H „ p h a s e p re c u r s o r s (le ak ag e)

In v e rte d H ex ag o n al P h a se H „

Figure 1.2.15 Interbilayer interactions.

In this introduction, only inter-conversions between the bilayer phase (La) and inverted hexagonal phase (Hu) will be discussed (Figure 1.2.15). The L« is arranged into extended bilayer sheets, whereas the Hn phase is characterised by long cylindrical cores set in a hexagonal hydrophobic lattice comprised o f the hydrocarbon c h a i n s . T h e transition o f lipids from the La to Hn phase usually occurs between -20 °C and 100 °C, and the temperature at which this occurs is referred to as the phase transition

temperature (Tm) Membrane fusion processes require lipids to depart from bilayer

aggregates. Ellens et al. observed that lipids with low Tm values participate more readily in this p r o c e s s . F u r t h e r m o r e , Cullis and De Kruijkk have correlated the molecular shape o f the lipid to the lipid aggregate phase preference:^^ for example, surfactants (which contain one alkyl chain and a polar head group) tend to form micelles, whereas phospholipids tend to adopt inverted hexagonal aggregates

1 Introduction Gene Therapy

Lipid Type Phase Molecule Structure

Surfactants and Detergents

Micellar Inverted Cone

Phospholipids such as Phosphatidylcholine (PC) Bilayer Cylindrical Phospholipids such as Phosphatidylethanol amine (PE) Hexagonal (Hn) Cone

Table 1.2.1 Lipid/water phases assembled by various lipids and the corresponding molecular shape of each lipid.

The molecular shape of lipids is affected by a variety of factors:

■ The effective head-group area which is determined by steric bulk, electrostatic repulsion, and H-bonding capacity.^’

■ The hydrocarbon chain which is affected by length and degree of saturation.^^

PE lipids have small head groups in comparison to the lipid chains, which gives the lipid a cone shape and as a result, these lipids prefer packing as a Hn phase.^ Conversely, phosphatidylcholine (PC) lipids have a increased phase transition temperature compared to PE lipids.^^ The introduction of the quaternary ammonium moiety increases the head-group size by affecting hydrogen bonding capacity of the lipid; PE lipids form H-bonds with neighbouring lipid molecules, whereas PC lipids

c a n n o t . Th i s results in PC lipids forming bilayer aggregates. Subsequently, Brown et

al. demonstrated that head group size, hydrophobicity and hydrogen bonding properties

of lipids increase Tm.^'

Intrafacial H-bonding also influences the phase transition temperature of the lipids.^^ For example, Eibl and Woolley demonstrated that intrafacial H-bonding can stabilise diacyl glyceride and phospholipid bilayers which leads to an increased Tm.^^

1 Introduction_______________________________________________________________ Gene Therapy

Reports suggest that the length of the hydrocarbon chain also influences the preferred phase adopted by the l i p i d . G e n e r a l l y , increases in the lipid chain length

cause the Hn phase to be adopted at lower temperatures. Seddon et a l observed that

increases in the chain length of PE lipids causes an decrease in Tm.^^ Furthermore, the introduction o f cw-olefin moiety caused a radical reduction in the phase transition temperature.^^ The preference for packing into the Hn phase has been attributed to the unsaturated hydrocarbon chains occupying a larger cross-sectional area, such that the hydrocarbon chains may be better accommodated within the Hn phase.^^ Furthermore, Binder and Gawrisch observed that the introduction of a cw-olefin in hydrocarbon chains resulted in a reduction in the half-thickness o f the lipid bilayers formed, compared to those which contain saturated lipid derivatives.^* This reduction in half­ thickness was accompanied by an increase in the mean cross-sectional area of the lipid.^* They attributed these differences to the degree o f rotational freedom of the saturated and unsaturated hydrocarbon chains. The cw-olefin moiety will have a torsion

angle of 120° whereas saturated chain will prefer trans 180° and gauche 60° rotational

angles.^* This results in hydrocarbon chains which contain a cz5-olefln moiety containing a “kink”, causing the chains to splay more widely.^^

Formulations for gene therapy often utilise mixtures of cationic lipids and phospholipids and the impact of cationic lipids on the structure adopted by

phospholipids has been determined.^^ Campbell et a l observed that the addition of the

cationic lipid DOTAP, to series of structurally related PC lipid derivatives, resulted in a decrease in the phase transition temperature and a reduction in the size of the liposome, which was independent of phospholipid chain length and the degree o f unsaturation.^^

Ellens and co-workers demonstrated that liposomes comprised of PE lipids

destabilize at or near the Tm of their lipid constituents.^^ Furthermore, Düzgünes et a l

reported that PE liposomes readily fuse with anionic lipids, whereas PC fuse poorly.^"* These studies suggested that the introduction o f minor structural changes within a lipid, can have significant ramifications for the aggregation properties o f the lipid, which in turn influences its biological behaviour.'*^’^^’^^’^'* The consequences for gene therapy are clear, the effectiveness and widespread utility o f the PE lipid DOPE,^^ suggests PE fusogenic properties are desirable for gene delivery.

1 Introduction Gene Therapy