Biomedical Engineering Reference
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Building on the success of iterative rational libraries, we
investigated whether smaller amphiphilic molecules would
effectively deliver small RNAs. To this end, cationic lipid-like
structures were chosen for three reasons. First, cationic lipids had
previously been shown to deliver siRNA.
5
Second, we hypothesized
that smaller cationic compounds would bind to highly anionic siRNA
tightly enough to prevent dissociation in the bloodstream, but
loosely enough to release the RNA once it was inside the cytoplasm.
Finally, we proposed that lipid tails — which facilitate interaction
with membranes, enhancing uptake by the plasma membrane and
destabilizing endosomal membranes
10
— might work in concert
with amines — which condense nucleic acids and induce endosomal
rupture through the proton sponge mechanism.
11
The pKa of amines
allows them to absorb protons without reducing endosomal pH. The
proton influx produces an ionic and osmotic gradient that causes
water to flow into the endosome, which eventually bursts.
12
To test this structural space, alkyl-acrylamide or alkyl-acrylate
lipid tails were conjugated to amine backbones via the Michael
addition, generating more than 700 first-generation lipidoids
13
(Fig. 7.2). Four structural parameters were modulated. First, alkyl-
acrylamide and alkyl-acrylate tail length was varied between 9 and
18 fully saturated carbons. Second, sundry amine backbones were
chosen in order to maximize structural diversity, thus increasing
the probability of identifying critical structural motifs; the chemical
space explored was, however, focused by knowledge obtained from
analysis of previous libraries. Third, the bond linking the amine
backbone to the alkyl tail was comprised of either a biodegradable
ester or a stable amide. Finally, the impact of backbone charge was
investigated by quaternizing amines with methyl iodide.
This
alkylating agent imparted a permanent positive charge on the
amine.
Once the library was synthesized, an efficient
11
assay was
required to screen the large number of structures. An ideal assay
would measure target and control gene expression concurrently,
so that potent silencing would not be confused with cytotoxicity.
To achieve this, human cervical cancer (HeLa) cells were made to
express stably both firefly and renilla luciferases (termed dual HeLa
cells). Lipidoids were complexed with firefly luciferase-targeting
siRNA (siFire) before the activities of both luciferases — which have
different substrates — were measured by luminescence the following
in vitro
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