0959 Specificity in Gene Silencing Using Multifunctional Nanoparticles.
Rao V. L. Papineni*#, Tao Ji#, Thirupandiyur S. Udayakumar, Mohammed M Shareef, Mansoor M. Ahmed, and Alan Pollack.
#Carestream Health, Inc., New Haven, CT and Department of Radiation Oncology, University of Miami, Miami, FL, USA
An efficient in vivo gene silencing therapeutics system utilizing the siRNA
technology requires the following criteria to be met- Ability to deliver high
volume/capacity of double stranded siRNA molecules to the site of interest, and to
efficiently transfer siRNA into the cells. siRNA delivery vehicle should also be capable
of protecting siRNA from the nucleases during systemic delivery. To address the
multiple prerequisites, we have developed nanoparticle-based delivery of siRNA, which
allows easy linking of siRNA, targeting antibodies, biologics such as cell penetrating
molecules, and other chaperones for gene silencing. We have utilized the high affinity
avidin-biotin interaction properties for this system. The biotinylated macromolecules
(siRNA, targeting and cell penetrating biologics) in precise stoichiometric concentrations
were linked to neutravidin decorated dye encapsulated polymeric nanoparticle
designated as High affinity hooker nanosphere (HAH NS), by simple incubation on ice.
Such a system also facilitates the simultaneous NIR fluorescence imaging in a
noninvasive manner along with targeted siRNA delivery. As a proof of concept, the
HAH-NS linked with biotinylated anti-PSMA antibody and MDM2 inhibiting
biotinylated siRNA was prepared. in vitro studies demonstrate the specificity in HAH
NS binding to PSMA expressing prostate cancer cells (LNCaP), and no binding to PC3
cells and fibroblasts which are negative for PSMA. Further, the anti-PSMA linked
HAH-NS significantly inhibited MDM2 in LNCaP-MST cells that have been
permanently transfected to over express MDM2 protein, suggesting the specificity of
targeted inhibition.
Ad -Luc
Necessity of Targeted Therapeutic System: Earlier studies
show that E2F1 overexpression works in concert with AS-MDM2 Ad -Luc+MM
and radiation therapy (RT) to enhance apoptosis and prostate
tumor growth inhibition. These studies involved intratumoral Ad -Luc+AS
injection with adenoviral-E2F1 and systemic delivery of AS-
MDM2, and thus not efficient in specific targeting of the tumor
Ad -E2F1
cells. Ad -E2F1+MM
Ad -E2F1+AS
Growth curves for
LNCaP tumors
treated with Ad-Luc,
Ad-E2F1, AS-MDM2
±RT for LNCaP and
PC3 tumors in nude
mice. The curves
represent the average
of numerical fits to the
individual growth
curves (n=16-17). The
combination therapies
were performed with
Ad-E2F1+AS-MDM2
±RT for LNCaP
tumors Serum PSA
(upper panel) and
Figure 1. Ad-E2F1, AS-MDM2 and Radiation Therapy
tumor volume were
Induced Tumor Regression in In-Situ Prostate Tumor estimated weekly.
Xenografts
Efficient Tool for
Targeted Therapeutic
System- in vivo targeted
gene-silencing methodology
capable of delivering
maximal amount of siRNA
at the target site.
Near IR dye encapsulate X-
SIGHT HAH-NS facilitates
the simultaneous near infra-
red fluorescence (NIRF)
imaging in a noninvasive
manner along with targeted
siRNA delivery.
Patent in process for Dr. Rao Papineni
Figure 2. Multifunctional X-SIGHT High-Affinity Hooker Nanosphere (HAH-NS)
*E.Mail: rao.papineni1@carestreamhealth.com
Figure 3. Cartoon describing the current work where biotin- siRNA-
Specificity in Gene Silencing
LNCaP-MST
MDM2 and biotin-anti-PSMA, the targeting antibody, was loaded to
(a)
the X-Sight High-affinity Hooker Nanosphere.
DAPI
Anti-MDM2
HAH
LNCaP-MST-IgG-Control-HAH-NS LNCaP-MST line that over express MDM2 were
treated with X-Sight 650 HAH-NS (Panels b,
HAH
HAH
MDM2-siRNA
(b) and c) for 24h. The MDM2 expression was
analyzed by immunofluorescence. Significant
Anti-MDM2
DAPI
reduction in the expression of MDM2 was
HAH
observed in LNCaP prostate cancer cell lines
only with targeting anti-PSMA antibody
HAH
conjugated X-SIGHT 650 HAH-NS (Panel “c”)
LNCaP-MST-Anti-PSMA-HAH-NS but not the control IgG antibody conjugated X-
HAH
MDM2-siRNA SIGHT 650 HAH-NS (Panel “b”).
(c)
HA
Anti-MDM2
H
DAPI
Figure 5. MDM2 siRNA loaded X-SIGHT-650 HAH-NS reduce the expression of
MDM2 in LNCaP cell line overexpressing MDM2 (LNCaP-MST).
Neutravidin a deglycosylated version of avidin, with a strong affinity
for biotin (kD10-15) is used here as a delivery tool for biotinylated
ligands. Simple incubation steps results in stoichiometric loading of
different functional components of the targeted siRNA delivery
system. Use of multi-tethered siRNA, and determination of its
advantages will be conducted in the successive studies that are
progressing.
Specificity in Targeting
LNCaP LNCaP/Fibroblast PC3 Fibroblast
Control IgG
IgG-HAH-NS
A B
DAPI
Figure 6 . Western Blot analysis demonstrates that MDM2 siRNA
loaded X-SIGHT HAH-NS reduce the expression of MDM2 in
Anti-PSMA-IgG-HAH-NS
Excitation Dye
LNCaP cells over expressing MDM2 (LNCaP-MST), only when
(650 nm)
the HAH-NS were with anti-PSMA targeting moiety.
Liver
DAPI
Tumor
Conclusions
X-SIGHT High-Affinity Hooker Nanosphere forms
an unique class of multifunctional nanoparticles
Figure 4. Biotinylated anti-PSMA antibody Targets X-SIGHT-761 HAH-NS to LNCaP Tumor (Left) and Cells that aid in specific targeting of tumor cells with
(Right). more than two molecular agents through minimal
conjugation procedures. X-SIGHT HAH-NS
in vivo NIRF Imaging: LNCaP cells were grown orthotopically in nude mice. At PSA levels 3-5ng/ml, NIRF imaging was
performed: Mice (A) was injected (i.v.) with IgG (nonspecific) conjugated HAH-NS and mice (B), with anti-PSMA potentially allows real-time monitoring of both the
conjugated HAH-NS. The anti-PSMA conjugated nanoparticles targeted the LNCaP tumor, indicating the specificity of the therapeutic delivery of the siRNA and the
anti-PSMA HAH-NS for LNCaP tumor cells. X-Ray and NIRF imaging using Kodak in-Vivo Multispectral FX system is regression of tumors. Current studies are in
as follows: X-Ray: 1 min exposure 2X2 binning, f-stop 2.8; Fluorescence: EX: 730; EM: 790; 1 min exp, no binning; f- progress to analyze the therapeutic efficacy of this
stop 2.8; signal contrasted for overlay. gene-silencing approach in orthotopic prostate
tumor model, we will also assess the advantages of
Targeting Prostate Cancer Cell Lines: Immunofluorescence demonstrating the specificity of Anti-PSMA X-SIGHT- 650
HAH-NS. Only LNCaP cells, that are PSMA positive, but not human fibroblast nor the PC3 cell, show binding and uptake using multi-tethered siRNA.
of anti-PSMA conjugated HAH-NS. Non-specific IgG-HAH-NS did not show binding to any cell type studied. Arrows
indicate the lack of anti-PSMA staining of fibroblasts in a fibroblast-LNCaP co-culture.
Carestream and X-SIGHT are trademarks of Carestream Health, Inc. The Kodak trademark is used under license from Kodak. Carestream Molecular
Imaging is a division of Carestream Health, Inc. Although the Kodak In-Vivo Multispectral Imaging System FX can be used for in vivo and in vitro
molecular imaging of materials, researchers should be aware that the methods of preparing and viewing the materials for molecular imaging may be
subject to various patent rights.
"Molecular Imaging - Wisdom To See For Maladies To Flee"
Dr. Rao V. L. Papineni