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siRNA Applications in Nanomedicine

I. Introduction
RNA interference (RNAi) is a method of regulating endogenous gene expression. RNAi is one of three mechanisms that can be used to silence a specific target gene, which functions by interfering with translation of mRNA and, subsequently, protein production. The RNAi process (illustrated in Figure 1) is initiated by the introduction of long, doublestranded RNA molecules (dsRNA) to a cell. Next, Dicer, a dsRNA-specific endoribonuclease, recognizes and cleaves these aberrant dsRNAs, producing short interfering RNA (siRNA). siRNAs are dsRNA molecules of 21 nucleotides with 3' overhangs of 2 nucleotides that form a ribonucleotide-protein complex known as the RNAinduced silencing complex (RISC). siRNAs are unwound at RISC and a single-stranded RNA molecule remaining attached to RISC binds to a complementary sequence on a target mRNA. Finally, the target mRNA is cleaved and degraded; thus, expression of the gene is interrupted. Although RNAi is a naturally occurring cellular process it is thought to function in defending the genome from transposons and viruses RNAi has been implicated in therapeutic gene silencing and the downregulation of disease-causing proteins.

Figure 1: An overview of the RNAi process. In addition to RNAi, antisense oligonucleotides (ODNs) and ribozymes can also be employed in gene silencing, however both methods are associated with significant drawbacks. ODNs are short, single-stranded DNA or RNA molecules that are complementary to target mRNAs such that hybridization to mRNA ultimately blocks expression of the target gene via mRNA cleavage. These results have only been demonstrated in vitro, as in vivo study has been impeded by difficulties associated with delivering ODNs. Furthermore, ODNs are less efficient and specific than siRNA in inhibiting gene expression. Ribozymes are RNA molecules with catalytic activity enabling

2 cleavage of single-stranded RNA, however ribozymes are only stable for a few minutes in serum. Therefore, RNAi and delivery of siRNA have become the focus of gene silencing applications.

II. Limitations to siRNA delivery

The challenges of the delivery of siRNA to its specific target mRNA in the specific cell/tissue are that siRNA may deviate from the target. The siRNA stability is a limitation in the delivery of naked siRNA from the blood to the target because the naked siRNA are small and can be filtered by the kidneys. The naked siRNAs can, also, be removed from the blood stream by endonucleases and exonucleases. The half-life of siRNAs can be increased from minutes to 14 days with modifications. The addition of a polymer, such as PBAVE, and other molecules, such as carboxy dimethylmaeic anhydride (CDM) linked with polyethylene glycol (PEG) or N-Acetylglucosamine (NAG), which cause the neutralization of siRNA. The addition of the polymer and other molecules enlarges and neutralizes the siRNA, which ultimately increases the half-life of siRNA. Another limitation in the delivery of siRNA is the aggregation of siRNA. siRNA is a positively charged particle and the positive charge hinders the delivery of siRNA because siRNAs clump with other siRNA. The positive charge, also, inhibits the siRNA binding to its specific target because siRNA binds to other negatively charged receptors instead of the target cell or tissue. When the siRNA is linked with PEG or NAG, the siRNA will be neutralized and it will not aggregate. A limitation is the siRNA targeting the specific cell or tissue; siRNA may target the specific cell by passive or active targeting. Passive targeting is when siRNA enters the cells because the target cancer cells have enhanced permeability from leaky vasculature, and the cancerous cells have a good retention of siRNA. However, passive targeting is not efficient and it is a slow method of targeting. Active targeting is the binding of siRNA ligand to the target receptor. Active targeting is an efficient method and active targeting is a quicker targeting method than passive targeting. The siRNA can have targeting ligands, which enable siRNA to bind to target receptors. Another limitation is the siRNA internalization because the target cell membrane is not permeable to siRNAs. siRNA is positively charged; since, the nanoparticle has a charge, the molecules of PEG and NAG neutralize the siRNA. Also, the nanoparticle has a large molecular weight; the siRNA cannot pass through the cell membrane. Therefore, the siRNA must go through the cell membrane by binding to the receptor and enters the cell by endocytosis. Another limitation is the endosomal escape of the siRNA into the cytoplasm. As the endosome matures, the pH of the endosome drops. This drop in pH causes CDM-PEG and CDM-NAG to be released from the nanoparticle. Therefore, the siRNA becomes positively charged and the PBAVE polymer on the siRNA lyses the endosome membrane. The contents of the endosome (siRNA) leak into the cytoplasm. Another limitation is the siRNA off-target effects, when the siRNA does not bind to the specific mRNA. A crucial step for gene silencing in RNA interference is when siRNA binds to mRNA. However, siRNA may not have exact sequence of nucleotides to bind to target mRNA, this could cause the siRNA to bind to other mRNA strands. The siRNA targeting can be enhanced by chemical modifications. The siRNAs have two single stranded RNAs; one of the strands is the guide strand, which binds to the mRNA, and the other strand is the passenger strand, which degrades and helps increase efficiency of targeting.

III. Approaches for siRNA delivery

Naked siRNA - Introduced into cellular compartment via manual hydrodynamic injection and electrophoresis to increase cellular permeability of naked siRNA - Early studies have shown some success in delivering naked siRNA; however, these methods are not ideal for in vivo applications. Direct conjugation to sense strand of siRNA Types of small molecules/peptides/polymers CPPs (Cell Penetrating Particles) PEG (Polyethylene Glycol) Cholesterol Long chain fatty acids Bile-salt derivatives Acid responsive polymers (contains PEG and NAG* targeting ligand) *NAG: N-acetylgalactosamine Results Increased gene transfer in vivo. Improved slicing of multiple genes in mice. Gene silencing to hepatocytes in vivo. Gene silencing in vivo.

- Results in smallest nanoparticles (less than 10nm in size), susceptible to enzymatic degradation Cationic Polymer Larger in size which is 100-300nm. It increases circulation time and allows specific tissue/cell interaction. Example, PEI (Polyethylenimine). Positively charges on the polymer (i.e PEI) electrostatically interacts with the negatively charges on the backbone of siRNA Polycation with positive surface charge is useful for in vitro gene transfer. However, when it is introduced in vivo via intravenous injection, it aggregates and forms larger structure by non-specific association with charged serum protein. It eventually leads to rapid clearance from the plasma. Therefore, polycation is undergone further chemical modification by conjugating blood-compatible functional polymers to enhance siRNA delivery efficiency. Examples of chemical modification: a. PEI-PEG b. Cyclodextrin-containing polycation c. Polylysine d. Natural polymer (such as chitosan) Cationic peptides (such as CADY and MPG-8) also complex siRNA efficiently. CADY:i. A secondary amphipathic peptide. ii. Has helical conformation in solution (regardless of pH). iii. Enters cell through mechanism which is independent of major endosomal pathway iv. Delivers siRNA into cytoplasm selectively and rapidly.

4 v. Promotes a significant siRNA associated knockdown of the target A novel CPP-modified protein was able to shield siRNA from degradation and deliver it to several target sites upon binding. Cationic Lipids Lipid vectors work more efficiently with siRNA than polymer vector due to its weak interaction with siRNA, which eventually leads to faster decomplexation in cytosol. Examples: Lipofectamine, Oligofectamine, Lipofectin, siPORT NeoFX and etc Only a limited number of cationic lipids used as carrier for in vivo siRNA delivery due to their poor colloidal stability and toxicity concerns. The first lipid formulations for in vivo siRNA delivery: 1,2-Dioleyl-3trimethylammonium propane (DOTAP) The chemical modification of solid lipid nanoparticle (SLN) (i.e low density lipoprotein(LDL)-mimicking nanoparticle) surface with PEG provides stabilization to SLN/siRNA-PEG polyplex micelles. Natural Liposomes Less than 200nm in size Least toxicity if compared to cationic polymers/lipids Unilammelar structure of liposomes (hydrophilic core & hydrophobic surfaces) protects encapsulated siRNA from degradation by surrounding RNases and facilitating internalization via membrane fusion Example of Drugs CALAA-01 Calando Pharmaceuticals leading drug candidate Is combination of RONDEL (RNAi/Oligonucleotide Nanoparticle Delivery) and a patented siRNA targeted M2 subunit of ribonucleotide reductases. The foundation of RONDEL is cyclodextrin-containing polymer Ribonucleotide reductase is essential in catalyzing conversion of ribonucleosides to deoxyribonucleosides and is needed for DNA synthesis and replication. Both siRNA and CALAA-01 shows anti-proliferative activity over multiple types of cancer cells. Is currently undergoing Phase I Trial at UCLA Medical Centre in Los Angeles. Interim result has demonstrated that CALAA-01 is well tolerated and has shown preliminary proof of RNAi activity in patient with high dose. Mediates specific gene inhibition as shown by mRNA and protein knockdown at tumor sites. Benefits of RONDEL More effective delivery: RONDEL binds to and self-assembles with siRNA in order to form uniform colloidal shaped siRNA (>100nm in size), which ease the accumulation at tumor sites. Increased stability: siRNA with RONDEL is more stable under physiological condition Fewer immune reaction: RONDEL allows repeating dosing without causing any immune response (such as interferon response that occurs in lipid delivery system)

5 Future: RNA interference can be a revolutionary method of therapeutic gene silencing. Triple negative breast cancer is when breast cancer cells dont have the estrogen, progesterone, and Her2/neu; this type of cancer is aggressive and less responsive to drugs. Mice had cancerous cells implanted into the mices mammary fat pad. The mice were injected with siRNA targeting triple negative breast cancer cells. The growth of cancerous cells was reduced and there was no metastasis (spread of cancer) into the lungs, liver, intestines and stomach.

Sample questions 1. Compare and contrast the three methods that can be used to silence specific target genes. Which one is the focus of therapeutic applications and why? 2. Describe two of the four delivery strategies discussed in the article (include any limitations if applicable). 3. Why is chemical modification of cationic polymers important?