Lipidoids: A Combinatorial Approach to siRNA Delivery - RNA Interference from Biology to Therapeutics - page 152

relationships from the previous library, this strategy requires knowledge of an effec-
tive starting point material for optimization and involves the undesirable use of
protection/deprotection chemistry.
A second approach was used to extend the utility of the first-generation lipidoid
library. Rather than modify existing core structures, scientists combined subopti-
mal-performing lipidoids to generate synergistic combinations [ 50 ] . Using a modest
starting library of 36 compounds, 630 binary pairs were generated and screened at
six weight fractions (0, 0.2, 0.4, 0.6, 0.8, 1.0), resulting in a total of 3,780 unique
formulations for a single siRNA concentration (20 nM) and lipidoid:siRNA (5:1)
weight ratio. A synergy value was defined for each formulation to quantify improve-
ments in gene silencing relative to the expected additive effects. It was confirmed
mechanistically that successful binary combinations could leverage the comple-
mentary abilities of the individual components. Specifically, it was observed that
one lipidoid promoted cellular uptake, while the second lipidoid promoted endo-
somal escape [ 50 ]. These in vitro data were supported by successful silencing
experiments in vivo. Interestingly, the lead combination influenced not only knock-
down but also biodistribution, enabling uptake by the liver and spleen, which had
not been observed using nanoparticles comprising either of the parent materials
alone. Notably, this combination approach is relevant to all classes of delivery mate-
rials. The efficacy of lipidoids may thus be further enhanced through combination
with other carrier types, such as polymers or siRNA conjugates.
While interesting, these two examples represent iterative extensions of the origi-
nal library; the application of creative chemistry can be used to generate disruptive
advances. A novel library was created through the efficient ring-opening of epox-
ides by amine substrates [ 49 ]. The resultant nondegradable amino alcohols con-
tained the key structural elements that had been previously defined—protonatable
amines and hydrophobic aliphatic tails—and showed vastly superior activity, pre-
sumably owing to the more stable linkage. The reaction ran to completion after
3 days, which is intermediate relative to the 1 day for acrylates and 7 days for acryl-
amides. A library of 126 lipid-like members was generated by parallel synthesis,
and structure-activity relationships were again evaluated. It was found that C14 was
optimal. Amine 113 ( N -methyl-2,2 ¢-diaminodiethylamine) was common to the two
top-performing materials in vitro. This amine, as well as the customly synthesized
amine 200, is very closely related to the original optimal backbone, amine 98, fur-
ther supporting the convergence of structure identified. Amine 200 was observed to
confer the greatest silencing ability in vivo, affording virtually complete silencing at
two orders of magnitude lower dose than other delivery systems.
This improved potency confers several advantages. First, it translates to less
siRNA and less formulation material being administered to patients, thereby increas-
ing tolerability. Second, it allows for the use of a larger dose than minimally required
efficacious dose, which was shown to increase the duration of silencing. Third, it
enables the administration of RNAi cocktails. The combination of siRNAs targeting
multiple genes would be useful for treating infectious diseases or multifactorial dis-
eases, such as cancer, in which multiple genes or pathways have been implicated. To
this end, it was shown that five hepatic genes could be simultaneously knocked down