In vivo delivery of RNAi by reducible interfering nanoparticles (iNOPs)

Article abstract of DOI:10.1021/ml4001003

RNA interference (RNAi) has considerable potential as a therapeutic strategy, but the development of efficient in vivo RNA delivery methods remains challenging. To this end, we designed and synthesized chemically modified interfering nanoparticles (iNOPs) composed of functionalized poly-l-lysine dendrimers modified with reducible spacers to facilitate release of small interfering RNAs (siRNAs) in vivo. We show that the novel siRNA-iNOP complexes mediate efficient gene-specific RNAi in cultured cells and in mice, where they display enhanced tissue-targeting capabilities. At a clinically feasible dose of 1 mg kg^-1, apolipoprotein B (apoB) siRNA-iNOP complexes achieved ～40-45% reduction of liver apoB mRNA and plasma apoB protein levels within 48 h of administration to mice, without apparent toxicity. Collectively, these findings demonstrate that siRNA delivery by the modified reducible iNOPs can provide a clinically significant and potentially tissue-specific new approach for RNAi therapy.

Full text of DOI:10.1021/ml4001003

Letter
pubs.acs.org/acsmedchemlett
In Vivo Delivery of RNAi by Reducible Interfering Nanoparticles
(
iNOPs)
Huricha Baigude, Jie Su,‡,∥ Joshua McCarroll,‡,⊥ and Tariq M. Rana*,†,‡,§
†,‡
†
Program for RNA Biology, Sanford−Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California
9
2037, United States
‡
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts
0
1605, United States
§
Department of Pediatrics and Biomedical Sciences Graduate Program, University of California, San Diego, California 92093, United
States
*
S Supporting Information
ABSTRACT: RNA interference (RNAi) has considerable
potential as a therapeutic strategy, but the development of
efficient in vivo RNA delivery methods remains challenging.
To this end, we designed and synthesized chemically modified
interfering nanoparticles (iNOPs) composed of functionalized
poly-L-lysine dendrimers modified with reducible spacers to
facilitate release of small interfering RNAs (siRNAs) in vivo.
We show that the novel siRNA−iNOP complexes mediate efficient gene-specific RNAi in cultured cells and in mice, where they
−1
display enhanced tissue-targeting capabilities. At a clinically feasible dose of 1 mg kg , apolipoprotein B (apoB) siRNA−iNOP
complexes achieved ∼40−45% reduction of liver apoB mRNA and plasma apoB protein levels within 48 h of administration to
mice, without apparent toxicity. Collectively, these findings demonstrate that siRNA delivery by the modified reducible iNOPs
can provide a clinically significant and potentially tissue-specific new approach for RNAi therapy.
KEYWORDS: Nanoparticle, siRNA delivery, iNOP, reducible nanoparticles
ene silencing by RNA interference (RNAi) is attracting
doses, and to deliver NOX2-specific siRNA to local arteries for
prevention of restenosis in an atherosclerotic rat model.
1
−3
19
G
increasing attention as a potential therapeutic strategy.
4
,5
During RNAi, small interfering RNA (siRNA) duplexes are
introduced into the cell and assemble in the RNA-induced
Here, we describe a novel form of iNOP designed to increase
the efficiency of in vivo siRNA delivery. We considered that
reducible nanoparticles may have advantages over non-
hydrolyzable materials by allowing siRNA to be released within
6
silencing complex (RISC), a multiprotein complex that cleaves
the complementary targeted mRNA or blocks its translation.
The sequence specificity of RNAi supports its potential to treat
diseases caused by gene products that are inaccessible to
traditional small molecule approaches and thus considered
nondruggable. However, effective siRNA-based therapies
require identification of target-specific siRNA sequences and
development of efficient vehicles to deliver siRNA to the
20−22
the cellular environment.
To test this, we modified iNOP-
7
by conjugating reducible spacers to the surface of the core
lipid-functionalized poly-L-lysine dendrimer. We prepared a
spacer (I) containing a disulfide bond (Scheme 1A) and
coupled it to iNOP-7 to give the new reducible derivative,
iNOP-7DS (Scheme 1B). Excess spacer I was included during
the coupling reaction to ensure highly substituted dendrimers
were obtained, which was confirmed by monitoring free amines
in the product by Kaiser’s test (data not shown), as well as
7
appropriate cells in vivo. Successful in vivo delivery of siRNA
has recently been reported using cholesterol-functionalized
siRNA and formation of stable nucleic acid−lipid siRNA
8
−11
particles (SNALPs).
Moreover, polymeric nanoparticles for
1
analysis by H nuclear magnetic resonance (NMR) and matrix-
RNAi have been successfully administered to humans,
providing encouragement for the development of RNAi-based
assisted laser desorption/ionization time-of-flight (MALDI-
TOF MS). As shown in Figure 1A, signals from the deprotected
spacer I (2.42, 2.79, and 2.95 ppm), the oleoyl group (0.75,
1
2−14
therapies.
We previously reported the design and synthesis of
interfering nanoparticles (iNOPs) as new siRNA delivery
1
.12, ∼1.75−1.85, and 5.21 ppm), and lysine residues (∼1.20−
1
1
5
1.54, 2.85, and 4.06 ppm) were observed in the H NMR
spectrum. iNOP-7DS showed a broad distribution in the
agents. iNOPs can be conveniently assembled by mixing
siRNA with functionalized poly-L-lysine dendrimers without the
1
6,17
need for complex liposomal formulation procedures
or
18
harsh covalent reaction conditions. One formulation, iNOP-7,
has been successfully used to deliver apolipoprotein B (apoB)-
specific siRNA in mice at therapeutically feasible and affordable
Received: March 14, 2013
Accepted: June 18, 2013
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ml4001003 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Scheme 1. Synthesis of Disulfide Bond-Containing Spacer I
the complex following a brief incubation (10 min at 37 °C)
with glutathione or dithiothreitol, indicating that the disulfide
bonds in iNOP-7DS are readily cleaved by reducing agents
a
(
A) and Reducible iNOP-7DS (B)
(
7
Figure 1C). Taken together, these data showed that iNOP-
DS is reducible and, given the physiological abundance of
glutathione (3−10 mM), the new iNOP derivative may be
expected to facilitate siRNA delivery by promptly releasing
siRNA upon entering the cell.
The electrostatic interaction between iNOP-7DS and nucleic
acids could yield a range of particle sizes. To examine this, we
measured the size distribution profiles of siRNA-iNOP-7DS
complexes by dynamic light scattering. At a weight ratio of 1:10
(
∼
N/P ratio 8:1), siRNA and iNOP-7DS formed nanoparticles
140 nm in diameter (Supplementary Figure 1), confirming
that the siRNA-iNOP-7DS nanoparticles are an ideal size for
a
Reagents and conditions: (a) acetic acid, EtOH, r.t., 4 h, 65%. (b)
23
EtOH, r.t., 6 h, 73%. (c) BOP reagent, DIPEA, DMF, 0 °C to r.t., 24 h,
application in drug or siRNA delivery.
8
4% then TFA, r.t., 30 min.
To determine whether iNOP-7DS could efficiently deliver
complexed siRNA to target cells in vitro, we tested silencing of
apoB transcription in FL83B mouse hepatocytes. ApoB is a
critical ligand for the low-density lipoprotein receptor and plays
an important role in cholesterol metabolism. For these
experiments, the stability of the apoB siRNA was enhanced
by chemical modification according to rules established in our
MALDI-TOF mass spectrum (Figure 1B). Major peaks were
seen at approximately m/z 9432.0, which corresponds to a
molecule with a degree of substitution 23, and minor peaks
were present at m/z 4730.9, corresponding to [M + 2H]. These
analyses confirmed we had successfully synthesized the highly
substituted iNOP derivative.
6
,7,24
previous studies.
FL83B cells were treated for 3 h at 37 °C
with iNOP-7DS complexed with apoB-specific or mismatched
siRNA, and apoB mRNA levels were then measured by
quantitative RT-PCR. As shown in Figure 2A, incubation of
cells with apoB siRNA-iNOP-7DS complexes effectively
silenced apoB expression (90% inhibition) compared with
cells treated with the control siRNA-iNOP-7DS (Figure 2A).
Cell viability was unchanged in the presence of iNOPs,
indicating that inhibition of apoB transcription was not due to
toxicity (Figure 2A). These results demonstrate that the
To test the ability of iNOP-7DS to release nucleic acid
following cleavage of the spacer, we mixed a 21-nucleotide
dsDNA with iNOP-7DS for 20 min at room temperature to
allow electrostatic association of the components. The complex
was then treated with reducing agents, and the products were
analyzed by electrophoretic mobility shift assay. We found that
the electrophoretic mobility of DNA was completely inhibited
when complexed with iNOP-7DS at a weight ratio of 1:6 (N/P
ratio 5:1) (Figure 1C). However, the DNA was released from
1
Figure 1. Characterization of iNOP-7DS. (A) H NMR spectrum showing signals from the disulfide-containing spacer, lipid chains, and lysine
residues. (B) MALDI-TOF MS analysis of iNOP-7DS. (C) DNA complexed with iNOP-7DS at 1:6 weight ratio (N/P ratio 5:1) was treated with or
without reducing agents at 37 °C for 10 min and then analyzed on a 2% agarose gel. (D) Schematic of iNOP-7DS reduction by glutathione (GSH)
or dithiothreitol (DTT).
B
dx.doi.org/10.1021/ml4001003 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 2. Efficient in vitro and in vivo delivery of siRNA by iNOP-7DS. (A) ApoB siRNA-iNOP-7DS complexes specifically silence apoB expression
in FL83B cells. Cells were treated for 3 h with iNOP-7DS with mismatched siRNA (m) or complementary siRNA (p). ApoB mRNA levels were
measured by qRT-PCR, and viability was assessed with the MTS assay. Results are expressed as percent of control cells and are the mean ± SD of
duplicate cultures from two representative experiments. (B) DDB1 siRNA-iNOP-7DS specifically silences DDB1 in HCT116 cells in the absence or
presence of serum. Cells were treated for 3 h in medium containing 0% or 10% FBS with control or DDB1 siRNA (50 nM) delivered by iNOP-7DS
(
N/P ratio 6:1) or Lipofectamine (5 μL per well for 6-well plate). DDB1 mRNA levels were measured by qRT-PCR and are expressed as percent of
control cells. (C) In vivo silencing of apoB by apoB siRNA-iNOP-7DS complexes. Mice were injected via the tail vein with PBS (mock) or with 2.0
−1
mg kg iNOP-7DS containing apoB-specific siRNA (pm) or mismatched siRNA (mm). Mice were sacrificed 48 h later and liver apoB mRNA levels
were measured by qRT-PCR. (D) ApoB protein levels are reduced in plasma of mice injected with apoB siRNA-iNOP-7DS. Animals were treated as
in panel B, and blood was drawn 24 h later. ApoB protein was analyzed in plasma samples by Western blotting. Fibronectin blotting was performed
as a protein loading control. Samples from two mice are shown. (E) Biodistribution of apoB guide strand siRNA in mouse tissues after i.v. injection
with iNOP-7DS complexes. Northern blots of total RNA (∼10 μg) isolated from the indicated tissues harvested 48 h after i.v. injection. Ethidium
bromide staining of tRNA is shown as a loading control.
reducible iNOP derivative is nontoxic and efficiently delivers
siRNA into cells.
siRNA complexes showed ∼40% decrease in liver apoB mRNA
levels (Figure 2C) and ∼45% decrease in plasma apoB protein
(Figure 2D). Moreover, Northern blot analysis of total RNA
isolated from the major organs of these mice showed the apoB
siRNA guide strand was present mainly in liver, spleen, and
lung at 48 h after injection (Figure 2E). These results showed
that iNOP-7DS complexed to chemically modified siRNA was
able to silence apoB expression efficiently in vivo. Remarkably,
apoB silencing with the new iNOP derivative required only 2.0
mg kg− siRNA, which is a therapeutically feasible dose when
scaled to humans. Of note, iNOP-7DS did not affect liver
enzymes and did not activate an immune response in mice
The stability of siRNA-containing nanoparticles may be
affected by serum components. To assess this, we incubated
human colon carcinoma (HCT116) and hepatocellular
carcinoma (HepG2) cell lines in 0% or 10% serum-containing
medium. Control or DDB1 siRNA was added to cells either
complexed with iNOP-7DS or in the presence of a standard
transfection reagent Lipofectamine 2000. Treatment of cells
with siDDB1-iNOP-7DS not only knocked down mRNA by
1
∼
90% in HT116 cells (Figure 2B) but also efficiently delivered
DDB1 siRNA to HepG2 cells, which are notoriously difficult to
(
data not shown).
Collectively, these findings demonstrate that siRNA delivery
transfect (Supplementary Figure 2).
Moreover, iNOP-7DS was as efficient as Lipofectamine 2000
in delivering siRNA into the cells (Figure 2B and
Supplementary Figure 2). Significantly, serum did not affect
DDB1 silencing in either cell type, indicating that iNOP-7DS is
stable in serum-containing medium.
by the novel reducible iNOP derivative may provide a clinically
significant new approach for RNAi therapy. Furthermore, the
derivatization strategy could be applied to create iNOPs for
tissue-specific RNAi therapies.
We next evaluated the efficiency of siRNA delivery by iNOP-
DS in vivo. For this, iNOP-7DS complexes were prepared
ASSOCIATED CONTENT
7
■
with chemically modified control or mouse apoB siRNA (N/P
*
S
Supporting Information
−1
ratio 8:1) and injected at 1−2 mg kg into wild-type C57BL/6
mice. Mice were bled after 24 h and sacrificed after 48 h.
Analysis of plasma and tissues from animals treated with apoB
Complete experimental details and supporting Figures S1−S3.
This material is available free of charge via the Internet at
http://pubs.acs.org.
C
dx.doi.org/10.1021/ml4001003 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(
12) Love, K. T.; Mahon, K. P.; Levins, C. G.; Whitehead, K. A.;
AUTHOR INFORMATION
Corresponding Author
(T.M.R.) Fax: +1 858 795 5328. E-mail: trana@
sanfordburnham.org.
Present Addresses
Boston Biomedical, Inc., 333 Providence Highway, Norwood,
■
Querbes, W.; Dorkin, J. R.; Qin, J.; Cantley, W.; Qin, L. L.; Racie, T.;
Frank-Kamenetsky, M.; Yip, K. N.; Alvarez, R.; Sah, D. W.; de
Fougerolles, A.; Fitzgerald, K.; Koteliansky, V.; Akinc, A.; Langer, R.;
Anderson, D. G. Lipid-like materials for low-dose, in vivo gene
silencing. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1864−1869.
*
∥
(13) Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.;
Cho, C. K.; Sah, D. W.; Stebbing, D.; Crosley, E. J.; Yaworski, E.;
Hafez, I. M.; Dorkin, J. R.; Qin, J.; Lam, K.; Rajeev, K. G.; Wong, K. F.;
Jeffs, L. B.; Nechev, L.; Eisenhardt, M. L.; Jayaraman, M.; Kazem, M.;
Maier, M. A.; Srinivasulu, M.; Weinstein, M. J.; Chen, Q.; Alvarez, R.;
Barros, S. A.; De, S.; Klimuk, S. K.; Borland, T.; Kosovrasti, V.;
Cantley, W. L.; Tam, Y. K.; Manoharan, M.; Ciufolini, M. A.; Tracy,
M. A.; de Fougerolles, A.; MacLachlan, I.; Cullis, P. R.; Madden, T. D.;
Hope, M. J. Rational design of cationic lipids for siRNA delivery. Nat.
Biotechnol. 2010, 28, 172−176.
Massachusetts 02062, United States.
Children’s Cancer Institute Australia Randwick, NSW 2031,
Australia.
Funding
This work was supported in part by grants from the NIH to
T.M.R.
Notes
⊥
The authors declare no competing financial interest.
(14) Davis, M. E.; Zuckerman, J. E.; Choi, C. H.; Seligson, D.;
Tolcher, A.; Alabi, C. A.; Yen, Y.; Heidel, J. D.; Ribas, A. Evidence of
RNAi in humans from systemically administered siRNA via targeted
nanoparticles. Nature 2010, 464, 1067−1070.
ACKNOWLEDGMENTS
■
We thank Craig Mello, Greg Hannon, Michael Czech, and
members of the Rana lab for helpful discussions and critical
reading of the manuscript.
(15) Baigude, H.; McCarroll, J.; Yang, C. S.; Swain, P. M.; Rana, T.
M. Design and creation of new nanomaterials for therapeutic RNAi.
ACS Chem. Biol. 2007, 2, 237−241.
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