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European Biopharmaceutical Review

The Rise of the Nano

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.


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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Surendra Nimesh received his MS in Biomedical Science and completed his PhD in Nanotechnology at the University of Delhi in India. After completing his postdoctoral research at École Polyetchnique of Montreal and Clinical Research Institute of Montreal Canada, he joined McGill University as a Research Assistant. Currently, he is working as NSERC postdoctoral fellow at Health Canada. He has authored several peer-reviewed research and review articles and book chapters and edited two books. His research interests include nanoparticles mediated gene, siRNA and drug delivery.
Surendra Nimesh
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