1.6 Preventing lipoplex degradation during endocytic trafficking
1.6.2 Endosome-destabilising devices
1.6.2.2 Proton sponge effect
In the 1980s, following elucidation of the mechanisms governing RME, several studies demonstrated that weak bases inhibit this process by disrupting endosome function (Wilemann et al., 1985). This observation was later explained by Behr (1997), who
hypothesised that the transfection efficiency of weakly basic polycations, in the absence of lysosomotropic agents, was due to their ability to circumvent degradation during endocytic trafficking by inducing rupture of the endosomal compartment.
Several polymeric carriers (Table 1.7) possess amino functions that exhibit buffering capacity within the endosomal range. When, as a consequence of endocytic uptake, such vectors reach the acidic environment of the endosomal compartment, their amino functions resist the proton pump-mediated change in pH by sequestering protons. Protonation of the vector results in an accumulation of positive charge within the endosome. This is accompanied by an influx of chloride ions, via membrane channels, in an attempt to maintain electroneutrality of the biological system. However, the accumulation of anions encourages excessive diffusion of water molecules, and causes swelling of the endosome. Once the osmotic pressure within the endosome rises beyond levels tolerable by the endosomal bilayer, it ruptures, releasing the vector to the cytosol. This phenomenon, summarised in Figure 1.17, is known as the proton sponge effect (Putnam et al., 2001).
38 Table 1.7: Non-viral vectors that exhibit intrinsic proton sponge capacity.
Gene carrier Reference
Polyethylenimine Akinc et al., 2005
Lipopolyamines Behr, 1997
Polyamidoamine dendrimers Pichon et al., 2010 Histidylated oligolysines Pichon et al., 2000
Figure 1.17: The proton sponge mechanism (adapted from Pathak et al., 2009).
While the use of certain polymers as gene transfer agents is often limited by cytotoxic effects, their proton sponge capacity has been profitably harnessed in combination with liposome- based strategies. As an illustration, Bandyopadhyay and colleagues (1998) have demonstrated the efficacy of second generation vectors, lipopolyplexes, in modulating PEI-induced toxicity in liver parenchymal cells. A targeted cationic liposome, formulated using DOTAP, DOPC
Vector
Lysis of endosome
Free vector Cl -
Cl -
Cl - Cl -
Cl - Cl - Extracellular
Intracellular
Endosome
39 and galactocerebroside, was used to encapsulate PEI/DNA complexes. This vector construct exhibited an approximate two-fold increase in exogenous gene expression in cultured human hepatocytes when compared to the transfection activity of the polyplex alone. However, in recent years, liposomes have been endowed with proton sponge capability by way of
incorporating pH-sensitive lipids in their formulations (Midoux et al., 2009). The discussion to follow pays attention to this emerging trend.
1.6.2.2.1 Imidazole-modified lipids
The amphoteric heterocycle, imidazole, has pKa of approximately 6.0 (Figure 1.18), which falls within the acidity range of the endosomal lumen. This aromatic compound is therefore able to induce rupture of the endosome via the proton sponge mechanism. Consequently imidazole and imidazole-containing compounds, notably the amino acid histidine, have received much attention in lipid design as a means of introducing endosome-destabilising functions into liposome formulations (Midoux et al., 2009).
Among the earliest attempts in this regard, is the design of a pH-responsive liposomal system; following the synthesis of three novel lipids, having tethered the imidazole ring to either cholesterol (Figure 1.19a), 1,2-dioleoyldeoxyglycerol or 1,2-dipalmitoyldeoxyglycerol as hydrophobic skeletons. Importantly, imidazole-conferred proton sponge capability was confirmed by a marked reduction in the in vitro transfection activity of such liposomes in the presence of bafilomycin A1, which inhibits endosomal acidification (Budker et al., 1996;
Midoux et al., 2009).
HN N HN NH
1
2 3 5 4
1
2 3 4 5
Figure 1.18: pH-dependent protonation of N3 of imidazole (C3H4N2) (adapted from Palleros, 2010).
pH > 6.0 pH < 6.0
40 In view of wide-spread interest in improving the efficacy of cationic liposomal gene transfer, the imidazole moiety has been applied to the preparation of a new class of helper lipids.
These lipids were constructed from a lipophosphoramidate skeleton, with a headgroup composed of either a histidine methyl ester (Figure 1.19b) or histidine residue. In vitro experiments revealed that the novel co-lipids enhanced the transfection capacity of
lipophosphoramide cytofectins, by a factor of up to 350, when combined in equimolar ratios.
Furthermore, these liposomal systems achieved levels of reporter gene expression superior to those designed using DOPE (Mével et al., 2008).
Alternatively, chemical elaboration has presented the possibility of integrating the proton sponge-inducing moiety within the cytofectin. As an example, Kumar and colleagues (2003) appended a single histidine residue to the headgroup of twin-chain cationic amphiphiles (Figure 1.19c). The combination of such lipids with twice the molar quantity of cholesterol demonstrated a hundred-fold greater transfection activity in the HepG2 cell line when
compared with two commercially available cationic liposome formulations. Furthermore, the fluorescence resonance energy transfer (FRET) technique was used to show that the histidine residue of the cationic lipids mediates membrane fusion within the pH range of 5 – 7 which, in addition to the proton sponge effect, contributes to endosome destabilisation and vector release. In a related study, a series of membrane-compatible amphiphiles was synthesised by the covalent grafting of multiple histidine residues to the hydrophilic end of a cationic
cholesterol derivative. While it was concluded that there is no linear relationship between the number of histidine residues in the headgroup and the transfection potential of this class of lipids; the introduction of liposomes prepared from mono- and tri-histidylated derivatives, in several cell lines, produced remarkably high levels of reporter gene expression in the
presence of up to 50 % serum (Karmali et al., 2006).
More recently, Shigeta and coworkers (2007) incorporated a histidine residue into an existing galactosylated cationic cholesterol derivative (Gal-C4-Chol), to afford a multifunctional lipid, Gal-His-C4-Chol (Figure 1.19d); which displayed integrated properties of hepatocyte-
specificity and endosomal pH-sensitivity. Liposomes formulated using the histidylated amphiphile, mediated significantly higher luciferase activity, in human hepatoma cells, than those formulated from the parent lipid, at all lipoplex charge ratios investigated.
41
N
N NH O
O
N
HN NH
O CH3 O
P O O
O R1
R1
NH N
H2N
NH O
NH CH2 CH2
R Cl- R
O OH
OH OH
HO
S NH
NH2
NH O
NH O N O
NH
Figure 1.19: Endosomal pH-sensitive lipids with imidazole groups, a) Cholesterol-(3- imidazol-1-yl-propyl) carbamate (ChIm) (Midoux et al., 2009); b) A neutral imidazole- lipophosphoramidate (Mével et al., 2008); c) L-histidine-(N,N-di-n-
hexadecylamine)ethylamide) (Kumar et al., 2003); and d) Gal-His-C4-Chol (Shigeta et al., 2007).
a)
R1 = C18H35
b)
c)
R = C15H31
d)
42