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the firefly luciferase signal was normalized to the Renilla luciferase signal. If neither
signal decreased, then the lipidoid tested was ineffective; if both signals
decreased, then the lipidoid tested was toxic. Only when the ratio of firefly-to-
Renilla luciferase decreased was the lipidoid of interest considered for subsequent
evaluation.
The first-generation library comprised more than 700 members. Several param-
eters were systematically varied to enable the determination of structure-activity
relationships among the lipidoids. First, the influence of the linker between the alkyl
chain and amine was evaluated. It was determined that the more stable amide bond
was preferred to the degradable ester bond. Second, it was demonstrated that the
postsynthetic introduction of a constitutive positive charge to the acrylate-derived
lipidoids by quaternization of the amine did not greatly improve efficacy. While
these insights were extremely valuable from a general perspective, more specific
information was required to elucidate the functionalities that conferred good trans-
fection ability among acrylamide-derived lipidoids.
This information was extracted from a heat map [ 18 ] [(see Fig. 7.3 ), which is a
useful tool for the interrogation of combinatorial libraries. The alkyl chain length
series ranged from C10 to C18, and the functional groups surrounding the backbone
amines were varied. It was observed that the top-performing materials were com-
posed of multiple primary or secondary amines separated by ethyl or propyl groups
(e.g., monomers 61-64, 95-103) and featured more than two short (<C12) tails.
These findings were unexpected and would likely never have been discovered using
the traditional rational approach, as standard cationic lipids generally have a lone
amine head and two longer (C18) tails [ 25 ] .
To test the hypothesis that critical common features had been elucidated, a sec-
ond-generation library of 500 lipidoids was synthesized. Screening of this library
confirmed that a convergence of structure had indeed been identified. Whereas only
3% of the first-generation library was able to confer knockdown comparable to or
greater than the positive control transfection reagent Lipofectamine2000™, more
than 50% of those examined from the second-generation library were able to achieve
this feat. Because multiple tails can be conjugated to oligoamines, the second-
generation library was synthesized using varied reaction stoichiometries to generate
lipidoids with a diverse number of tails. It was found that retention of one unreacted
secondary amine—that is, having one tail less than complete substitution—was
most favorable. Notably, many of the effective lipidoids were structurally different
from both conventional lipids and cationic polymers. This set substantially expanded
the collection and chemical diversity of materials known to facilitate siRNA delivery
into cells.
To confirm that lipidoids could transfect multiple cell types, the top-performing
materials were tested in the human hepatocellular carcinoma cell line HepG2 and
primary bone marrow-derived murine macrophages at multiple doses in vitro.
Interestingly, the lipidoids displayed variable efficacy in the different cell types.
Silencing was consistently observed to be dose dependent, indicating that the
dynamic range under investigation was nonsaturating. While the overall silencing at
low lipidoid-siRNA complex concentrations in HeLa and HepG2 cells lines is less
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