Biomedical Engineering Reference
In-Depth Information
produced specifically by hepatocytes, delivery is not confused with
that to kupffer cells, immune cells that reside in the liver.
13
Lipidoids were mixed with cholesterol and PEG-lipid conjugates
prior to intravenous injection. These excipients had been shown
to increase serum stability, resistance to aggregation, and
in vivo
15, 16
tolerability.
ingredients was
also shown to influence biophysical characteristics,
The molar ratios of the three
in vivo
in vivo
potency,
17
and tolerability;
more details relating to the importance of
formulation will be presented later in this chapter. After administering
a dose of 5 mg/kg, 5 of the 17 compounds silenced target protein
expression by over 50%, with one (termed 98N
) silencing F7 by
more than 90% relative to saline control. Before continuing with
additional
12
,
whose reaction yields multiple products. The chemical mixture was
separated into constituents that were distinguished by the number
of tails attached to the amine backbone. A structure with five tails
(98N
in vivo
models, we discerned the active isomer of 98N
12
-5), one less than saturating substitution, was identified as
the active component, while the other variants were found to be
relatively ineffective.
Purified 98N
12
-5 was tested to determine how much siRNA
was required to silence a target gene by 50%, how long silencing
persisted, whether F7 silencing resulted in the expected physiological
effect, and whether it could mediate the silencing of another liver-
specific gene, Apolipoprotein B (ApoB). 98N
12
-5 was shown to result
in dose-dependent silencing that lasted more than two weeks. The
silencing of F7 and ApoB led to physiologically relevant outcomes,
namely increased clotting time and decreased LDL levels. Finally,
the compound was evaluated in rats, which are used for pre-clinical
immunological assays, and in non-human primates. It potently
silenced endogenous target genes in both species.
12
-5 was selected from a library that varied the length of
a fully saturated alkyl tail attached to amine backbones.
98N
12
13
Since
alkyl tail length was found to influence delivery dramatically, we
explored whether other structural changes to the hydrophobic
tails might impact efficacy.
18
Amine backbones 98 and 100 from
the original lipidoid library were conjugated to 17 acrylates with
oxygens, branched carbons, or amines in the tail. Experiments
using dual HeLa cells confirmed that
in vitro
silencing was affected
by tail structure. Conjugating PEG (CH
O) directly to the amine
backbone inhibited silencing, while attaching PEG-like repeats (for
example, CH
CH
2
2
CH(CH
)O or CH
CH
CH
O)
resulted in compounds that
2
3
2
2
2
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