Lipidoids: A Combinatorial Approach to siRNA Delivery - RNA Interference from Biology to Therapeutics - page 148

Fig. 7.4 The structures of 98N 12 -5(1), the five-tailed isomer of triethylenetetramine-laurylamino-
propionate with a free internal amine, cholesterol, and mPEG 2000 -C14 glyceride (reproduced with
permission from [ 31 ] )
In addition to formulation composition, the effects of siRNA loading, particle
PEGylation, and particle size on in vivo delivery efficacy were investigated [ 31 ] .
Loading is an important consideration because, for a given dose of siRNA, a higher
siRNA loading translates to a lower dose of administered lipid. Decreasing the dose
of lipid in vivo could decrease toxicity; however, it could also decrease efficacy. It
was determined that maximal siRNA loading occurs at a lipid:siRNA of ~7.5:1
(wt:wt). A comparison of efficacy and tolerability at multiple lipid:siRNA loading
ratios confirmed that this minimum amount of lipid required to entrap the entire
siRNA dose was optimal.
Small changes in the anchor chain length of PEG-lipids were observed to affect
efficacy significantly. Pharmacokinetics are strongly influenced by the extent of
particle PEGylation and the rate at which deshielding occurs [ 32 ] . The lipid portion
of PEG-lipids is incorporated into lipid particles hydrophobically rather than cova-
lently, so this deshielding rate can be controlled by altering the length of the alkyl
chain lipid anchor [ 33 ]. To determine the optimal length of the hydrophobic lipid
tail, a glycerol backbone was derivatized with one PEG chain (MW 2000) and two
alkyl chains of various lengths, ranging from C10 to C16. It was found that C14
lipid anchor length maximized the combination of desirable efficacy and tolerabil-
ity. Next, the effect of particle size on the performance of this formulation was
examined. Particles were extruded through filters with pore sizes ranging from 50
to 150 nm, and it was confirmed that smaller particles increase efficacy.
The final optimized formulation (see Fig. 7.4 ) had a lipid composition of lipidoid
98N 12 -5(1):cholesterol:PEG-lipid=42:48:10 (mol:mol:mol), total lipid:siRNA=
~7.5:1 (wt:wt), C14 alkyl chain length on the PEG-lipid (MW 2000), and a mean
particle size of ~50-60 nm. The surface charge was measured to be~neutral
(~+3 mV). The particles effectively protected the entrapped siRNA, increasing its