Biomedical Engineering Reference
In-Depth Information
These structure-function relationships were used to inform the
synthesis of a second-generation lipidoid library, consisting of more
than 500 new molecules. Of the 1,200 total structures, ~5% silenced
target gene expression at least as efficiently as the positive control,
Lipofectamine 2000, a commercially-available transfection reagent.
The second-generation library led to the insights that compounds
with amide linkages, more than two alkyl tails between 8 and 12
carbons, and one alkyl tail less than saturating substitution delivered
siRNA to dual HeLa cells most efficiently. After identifying effective
candidates in HeLa cells, the ability of compounds to silence genes
in human hepatocellular carcinoma (HepG2) cells and primary
bone marrow-derived murine macrophages was investigated. These
additional
assays were performed because transfection
efficiencies can vary dramatically between different cell lines.
Lipidoids were less proficient than Lipofectamine 2000 when
silencing endogenous genes in HepG2 cells but more efficient when
silencing endogenous genes in macrophages, with 50% target gene
reduction at siRNA doses as low as 1 nM. By contrast, Lipofectamine
did not silence macrophages even at 10 nM, suggesting lipidoids
might be useful for transfecting cell lines that were previously
refractory to treatment.
in vitro
13
7.4
Translation: Moving from
in vitro
to
in vivo
Screening
Based on the results of
in vitro
screens in multiple cell lines, 17
compounds were chosen for
in vivo
evaluation. Although
in vitro
results do not exactly correlate to
results, they do identify
a subset of materials that should be tested in animals, thereby
saving both time and money. A desirable
in vivo
screen will measure
gene expression of a target produced specifically by one cell type
quickly and inexpensively. To this end, we measured hepatocyte
delivery by targeting Factor 7 (F7). Hepatocytes were chosen as a
target cell because they play an important role in cancer, hepatitis,
hypercholesterolemia, malaria, and many other pathologies.
in vivo
14
F7
was selected as a target gene, since it is found in plasma and has a
short half-life. This means F7 levels can be measured in blood serum
without sacrificing the mouse, and knockdown at the protein level
can be quantified within hours of injection. Moreover, since F7 is
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