Biology Reference
In-Depth Information
than with commercial reagents, lipidoids provide for more effective silencing in
primary bone marrow-derived macrophages. The library approach herein described
provides a substantial number of new materials that may be useful for transfection
of cells that have thus far proven refractory to transfection by currently available
commercial reagents.
7.5
Formulation Considerations
The top-performing materials were next evaluated for the ability to confer silencing
in vivo as the objective of the work was to discover and develop carriers for thera-
peutic applications. The liver represents a desirable target organ, as it is well fenes-
trated and well perfused. Successful delivery to hepatocytes would enable RNAi to
address sundry diseases, including dyslipidemia, fibrosis, hepatitis, and hepatocel-
lular carcinoma.
Since the intended target tissue was liver parenchyma, customized formulation
was important as cationic lipid formulations often accumulate not only in the liver
but also in the lungs and spleen [
26
]. Moreover, oftentimes the majority of liver-
associated material is taken up by components of the mononuclear phagocyte system
(MPS) (Kupffer cells) of the liver rather than by the parenchyma (hepatocytes).
Factor VII was selected as an endogenous reporter as it is produced exclusively
by hepatocytes. This protein is secreted into the blood. As a consequence, knock-
down can be studied longitudinally by taking serial blood draws. This decreases the
number of animals required to conduct a given study and eliminates the need to
sacrifice animals to obtain tissue for analysis, thereby obviating the necessity to
compare data collected from different animals over a time course. Additionally, fac-
tor VII has the shortest half-life among the clotting factors (2-5 h), so silencing at
the mRNA level is manifest as silencing at the protein level with minimal lag.
Simple ionic complexes are generally not suitable for systemic administration
owing to poor serum stability, a tendency to form aggregates, and poor tolerability
[
27
]. Specifically, excess charge is associated with nonspecific interactions with bio-
logical surfaces, increased protein binding, opsonization, rapid clearance by the
MPS, hemolysis, and cytotoxicity [
28
]. For these reasons, lipidoids were formulated
with cholesterol and polyethylene glycol (PEG)-lipid. These excipients were
selected because they had previously been shown to stabilize lipid nanoparticles
[
29
]. Cholesterol is naturally found in lipid membranes, providing structure to bilay-
ers by occupying the space between lipid tails. PEGylation prevents aggregation,
decreases uptake by the MPS, and increases circulation time. Inclusion of PEG-lipid
in the formulation was critical, as it resulted in the formation of small, nonaggre-
gated particles that were accessible to hepatocytes via 100-150 nm-sized endothe-
lial fenestrae [
30
]. Literature values for molar ratios of cholesterol (30-50%) and
PEG-lipid (5-10%) were used as starting points. The lipid composition was system-
atically optimized to achieve a robust formulation with high in vivo activity.
Search WWH ::
Custom Search