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European Biopharmaceutical Review
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Nanomedicine is becoming an attractive candidate for effi cient siRNA
delivery methods, as nanoparticles emerge as therapies for anti-tumour
treatments. However, a number of issues must be resolved before it can
be used as a new model
RNA interference (RNAi) has been extensively employed to knockdown
the expression of malfunctioning genes and to defi ne gene function in
mammalian cells. It is a highly conserved natural process that controls
the selective post-transcriptional down-regulation of target genes in
the cells. RNAi is mediated through small sequences of 21 to 23
nucleotides in double-stranded RNAs, referred to as siRNAs (small
interfering RNAs). These siRNAs possess capability to degrade mRNAs
(messenger RNAs) that are complementary to one of the siRNA strands.
Fire et al were the first to report that the double-stranded RNA
molecules (dsRNA) mediates gene silencing (knockdown of expression) in
Caenorhabditis elegans (1). Gene silencing bears several advantages over
conventional chemotherapy in the treatment of cancer, which is
characterised by uncontrolled growth of cells, invasion and metastasis,
such as reduced non-specifi c tissues toxicity and high-target specifi
city. However, the therapeutic application of RNAi is limited due to the
poor cellular uptake and rapid nuclease degradation of siRNA molecules.
Hence, a delivery vehicle is highly desirable which can administer
siRNA effi ciently, safely and repeatedly to in vitro and in vivo
milieu.
The Wider Scale
Nanomedicine is a broader term which includes: polymeric micelles;
quantum dots; liposomes; polymer-drug conjugates; dendrimers;
biodegradable nanoparticles; inorganic nanoparticles; and other
materials in nanometer size range with therapeutic relevance. It has
been suggested that nanoparticles are one of the candidates with the
most potential in nanomedicine, with applicability in targeted drug and
gene delivery. Furthermore, due to their small size, nanoparticles
provide better tissue penetration and targeting.
Nanoparticles prepared from cationic polymers have been extensively
reported to deliver DNA and siRNA. These nanoparticles can be
sub-divided into nanospheres – spherical nanometer size particles where
the desired molecules can either be entrapped inside the sphere, or
adsorbed on the outer surface, or both. Nanocapsules have a solid
polymeric shell and an inner liquid core, and the desired molecules can
be entrapped (see Figure 1) (1,2). Numerous natural and synthetic
polycationic polymers including chitosan and polyethylenimine (PEI) have
been utilised for preparing nanoparticles to deliver siRNA.
Basic Principles and Mechanisms
The process of gene silencing mediated via RNAi takes place in the
cytoplasm of the cells (see Figure 2). RNAi is a multi-step process
which initiates with the binding of dsRNA-specific endonuclease (a
cytoplasmic ribonuclease III (RNase III)-like protein) called Dicer,
which is capable of cleaving long dsRNAs. Binding of dicer is followed
by cleavage of long dsRNA into smaller duplexes with 19 paired
nucleotides and two nucleotide overhangs at both 3’-ends. The small
dsRNAs generated are called siRNAs. The siRNA molecules bind to a
nuclease containing a multi-protein complex called RNA-induced silencing
complex (RISC).
The RISC complex, by virtue of its RNA helicase activity, unbinds
the double stranded siRNA molecule, removing the sense strand, which is
further degraded by cellular nucleases. The antisense strand of siRNA
remains bound to the RISC complex and is directed to the target mRNA
sequence, where it anneals complementarily by Watson-Crick base pairing.
Finally, the RISC complex cleaves the target mRNA by endonucleolytic
activity, thereby preventing its translation into protein. At a later
stage, the cleaved products are released and degraded by cellular
nucleases, leaving the unengaged RISC complex to further search for more
target mRNAs and carry out its activity of interference (2).
Advantages and Limits
Nanoparticles derived from polymers possess several advantages such
as: ease of manipulation with scope to change the molecular weight (MW);
geometry (linear and branched); stability; safety; low cost; and high
flexibility regarding the size of transgene delivered. Nanoparticles can
be easily tagged with various targeting moieties, such as RGD peptides
or transferrin, to achieve targetspecific siRNA delivery (3,4).
Furthermore, owing to its size (usually 10-200nm), nanoparticles can
readily interact with surface biomolecules inside the cell. Also, due to
their small size, nanoparticles can penetrate tissues such as tumours
in depth with a high level of specificity, thereby improving the
targeted delivery of drug or gene (5).
Despite being so advantageous and successfully implicated in
numerous studies, polymeric nanoparticles have some limitations. The
cationic polymers, constituting nanoparticles, undergo strong
electrostatic interaction with plasma membrane proteins, which can lead
to destabilisation and ultimately rupture the cell membrane (6).
Strategies based on the reduction of surface charge by coating with
anionic or neutral molecules such as hyaluronic acid (HA) or
polyethylene glycol (PEG) have been investigated to alleviate this issue
(7,8).
siRNA Delivery Reception
Although initial studies reported delivery of ‘naked’ siRNA, later
use of lipids, polymers, specific peptides and various other
nanoparticles made rapid progress in the evolution of therapeutic
potential of siRNA. Successful in vitro and in vivo delivery studies of
DNA by chitosan and PEI makes them attractive candidates for siRNA
delivery (3,9).
Chitosan is one of the most widely investigated non-viral, naturally
derived polymeric gene delivery vector, comprising
N-acetyl-D-glucosamine and β (1, 4)-linked D-glucosamine units. The
transfection efficiency of chitosan/ DNA nanoparticles depends on
several factors such as: the degree of deacetylation (DDA) and MW of the
chitosan; pH; protein interactions; N/P ratio (ratio of nitrogen of
amine in polymers to phosphate in DNA); cell type; nanoparticle size;
and interactions with cells (10).
Katas et al seems to be the first group to report the use of
chitosan to deliver siRNA in vitro (11). The studies done on two
different cell lines, CHO K1 and HEK 293, revealed that the method of
siRNA association with chitosan plays an important role on gene
silencing. Nanoparticles of chitosantripolyphosphate entrapping siRNA
were found to be better vectors in comparison to chitosan-siRNA
complexes, possibly due to their high binding capacity and loading
efficiency.
Howard et al engineered novel chitosanbased siRNA nanoparticles which
mediated knockdown of endogenous enhanced green fluorescent protein
(EGFP) in both H1299 human lung carcinoma cells and murine peritoneal
macrophages (77.9 per cent and 89.3 per cent reduction). Significant RNA
interference was observed after nasal administration of chitosan/siRNA
formulations (37 per cent and 43 per cent reduction) in bronchiole
epithelial cells of transgenic EGFP mice (9).
In a recent study, aerosolised chitosan/ siRNA nanoparticles used to
dose transgenic EGFP mice showed significant EGFP gene silencing (68 per
cent reduction compared to mismatch group) (12).
PEI (2-800kDa MW) is one of the most extensively investigated gene
carriers due to its high membrane destabilisation potential and charge
density (nucleic acids condensation capability) (13,14). In one study,
an ionic complex of branched PEI (MW 750 kDa) and alginic acid was
prepared to amalgamate the properties of cationic PEI with
polysaccharide alginate (15). The PEIalginate (6.26 per cent amino
groups derivatised by alginate) nanoparticles efficiently delivered
siRNAs into mammalian cells, resulting in an 80 per cent suppression of
Green Fluorescent Protein (GFP) expression.
In another published study, nanoparticles of PEI were prepared by
acylating PEI with propionic anhydride followed by cross-linking with
polyethylene glycolbis( phosphate) (16). In vitro siRNA delivery studies
in HEK 293 cells with nanoparticles showed up to 85 per cent inhibition
of GFP gene expression, which was almost equal to that for
siRNA-Lipofectin (81 per cent inhibition).
Future Perspectives
The successful implication of RNAi depends on several parameters
including: siRNA protection; high transfection efficacy; reduced
toxicity and absence of non-specific effects; high potency even at small
amounts of siRNAs; adaptation to various treatment regimens; and
efficient vectors to bypass intracellular and extracellular barriers to
reach their target tissue or organ.
So far, nanoparticles have been successful in the delivery of siRNA for
anti-tumour treatments in different experimental models. Despite
excitement from a large number of animal model studies, including
systemic delivery to non-human primates, there are a number of issues
that must be overcome before RNAi could be harnessed as a new
therapeutic modality (17). The ideal nanoparticles for a siRNA carrier
system should achieve long circulation time, low immunogenicity, good
biocompatibility, selective targeting and efficient penetration to
barriers, such as the vascular endothelium and the blood brain barrier,
self-regulating release without any clinical side-effects.
References
1. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE and Mello CC,
Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegans, Nature 391: pp806-811, 1998
2. Leung RKM and Whittaker PA, RNA interference: from gene silencing to
gene-specific therapeutics, Pharmacology & Therapeutics 107:
pp222-239, 2005
3. Schiffelers RM et al, Cancer siRNA therapy by tumor selective
delivery with ligand-targeted sterically stabilized nanoparticle,
Nucleic Acids Res 32: e149, 2004
4. Davis ME, The first targeted delivery of siRNA in humans via a
selfassembling, cyclodextrin polymer-based nanoparticle: from concept to
clinic, Mol Pharm 6: pp659-668, 2009
5. Cuenca AG, Jiang H, Hochwald SN, Delano M, Cance WG and Grobmyer SR,
Emerging implications of nanotechnology on cancer diagnostics and
therapeutics, Cancer 107: pp459-466, 2006
6. Fischer D, Li Y, Ahlemeyer B, Krieglstein J and Kissel T, In vitro
cytotoxicity testing of polycations: influence of polymer structure on
cell viability and hemolysis, Biomaterials 24: pp1,121-1,131, 2003
7. Jiang G et al, Hyaluronic acidpolyethyleneimine conjugate for target
specific intracellular delivery of siRNA, Biopolymers, 89: pp635-642,
2008
8. Duan Y, Yang C, Zhang Z, Liu J, Zheng J and Kong D, Poly(ethylene
glycol)- grafted polyethylenimine modified with G250 monoclonal antibody
for tumor gene therapy, Hum Gene Ther 21: pp191-198, 2010
9. Howard KA et al, RNA interference in vitro and in vivo using a
chitosan/ siRNA nanoparticle system, Molecular Therapy 14: pp476-484,
2006
10. Mansouri S et al, Characterization of folate-chitosan-DNA
nanoparticles for gene therapy, Biomaterials 27: pp2,060-2,065, 2006
11. Katas H and Alpar HO, Development and characterisation of chitosan
nanoparticles for siRNA delivery, Journal of Controlled Release 115:
pp216-225, 2006
12. Nielsen EJ et al, Pulmonary gene silencing in transgenic EGFP mice
using aerosolised chitosan/siRNA nanoparticles, Pharm Res 27: pp2,520-
2,527, 2010
13. Godbey WT, Wu KK and Mikos AG, Poly(ethylenimine) and its role in gene delivery, J Control Release 60: pp149- 160, 1999
14. Creusat G et al, Proton sponge trick for pH-sensitive disassembly of
polyethylenimine-based siRNA delivery systems, Bioconjug Chem 21:
pp994- 1002
15. Patnaik S et al, PEI-alginate nanocomposites as efficient in vitro
gene transfection agents, Journal of Controlled Release, 114: pp398-409,
2006
16. Nimesh S and Chandra R, Polyethylenimine nanoparticles as an
efficient in vitro siRNA delivery system, Eur J Pharm Biopharm 73:
pp43-49, 2009
17. Zimmermann TS et al, RNAi-mediated gene silencing in non-human primates, Nature 441: pp111-114, 2006
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