Introduction:
Rebalancing the RANKL/OPG-system is an effective treatment-strategy for osteoporosis.
Here we used virus-like particles (VLPs) as a small RNA based gene delivery vehicle
to inhibit RANKL expression. In our former studies we showed a significant short-term
knockdown of RANKL by VLP mediated RNA-interference via weekly i.p. injection. Chitosan
is a promising possibility for prolonged (up to 21 days) drug delivery in vivo. In
the present study we investigated the effects of RANKL-knockdown by chitosan embedded
siRNA-VLPs.
Methods:
VLP production and loading VLPs were produced in Hi5 insect cells and purified via
size-exclusion chromatography. VLPs were loaded with desired target drug (e.g. expression
cassette for GFP or RANKL siRNA) and incubated for 30 min at room temperature. Reassembly
was performed in 5 L HEPES buffer containing CaCl2 for capsid formation at 4 °C overnight.
Chitosan gel production For chitosan gel production 190 mg of Chitosan (95% deacetylated,
Heppe Medical Chitosan GmbH, Germany) was dissolved in 10 mL acetic acid (0.1 M).
The solution was cooled to 4 °C and ß-Glycerophosphate (3 g) was added slowly. The
final liquid was clear and homogeneous and stored at 4 °C until usage. In vitro validation
For in vitro applications precooled chitosan gel was mixed with GFP-cDNA loaded VLPs
and transferred into transwells (400 µm pore size). Transwells were inserted into
prepared 24well plates containing human cell lines (osteoblasts or embryonic kidney
cells). GFP expression was documented daily up to 14 days. Furthermore, we tested
VLP stability and release under physiological conditions by dot blot analysis. In
vivo application All animal procedures were approved by the local Animal Care and
Use Committee and performed according to the German animal protection laws. 3-month-old
female Sprague-Dawley rats were used for VLP injection (Janvier Lab, France). Chitosan
embedded VLPs were i.p. injected Tibia, lumbar vertebrae and blood samples were removed
for analysis.
Results:
VLP release was detected for 14 days through fluorescence microscopy and for 21 days
by dot blot analysis. The in vivo experiments showed a significant and time depending
knockdown of RANKL. 3 Days after VLP injection we detected a RANKL knockdown by 43%.
This knockdown was even increased after 14 days (67%). Knockdown was superior to former
studies with VLP mediated RNA interference without chitosan. Toxic effects or inflammations
were not observed in vivo.
Discussion:
In this study we demonstrate a successful transduction of chitosan embedded VLPs in
vitro and in vivo. A significant knockdown of RANKL expression 3 and 14 days after
injection in vivo indicates a long time function of VLP-encapsulated siRNA. This improved
effect of chitosan embedded VLPs suggests it as a promising tool for specific siRNA
therapy.
