The recent discovery of ribonucleic acid interference (RNAi) has opened up an entirely novel and exciting field of therapeutic drug design. Since its discovery by Fire et al. in 1998, this Nobel prize winning technology has rapidly emerged as a promising new tool for various fields. The potential for RNAi in treating multiple diseases has become the focus of a large number of academic laboratories and pharmaceutical companies around the world.
Relative low cost of production, less toxicity, high potency and ease of synthesis make small interfering ribonucleic acid (siRNA) a preferred therapy. Additionally, the use of siRNA technology has now become a hallmark for validation of basic science observations and unknown gene function determination. However, due to its large molecular weight (~13 kDa) and polyanionic nature (~ 40 negative phosphate charges), naked siRNA does not freely cross the cell membrane. Hence, delivery systems are required to facilitate its access to its intracellular sites of action.
Various non-viral delivery vehicles for siRNA
Ligand targeted sterically stabilised nanoparticles: Self-assembling nanoparticles with siRNA have been constructed with polyethyleneimine (PEI) that is polyethyleneglycolated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG).
Polyethyleneimine possesses a high cationic charge density due to a protonable amino group and hence has ability to condense siRNA into complexes. The PEG moieties form a protective layer that shields the polyplex core, giving rise to the steric stabilisation of the polyplex structure against undesirable interactions with surrounding environment. RGD enables effective targeting.
Inside the cells, these nanoparticles are entrapped by the endosomes. But PEI possesses membrane disruptive properties. It swells and bursts the endosomal membrane through protonation of excess amine groups. This 'proton sponge' effect causes influx of chloride ions, which induces osmotic swelling and subsequent disruption of the endosome. The release of siRNA from polyplex is aided by the association of cellular anionic lipids with the cationic polymer to form a neutral ion pair. Formation of these ion pairs reduces the electrostatic interaction between the negatively charged nucleic acid with the delivery vehicle, thereby enabling nucleic acid release.
Polymer brush stabilised polyplex for a siRNA carrier: Cationic comb-type copolymer (CCC) possessing a polycationic backbone (less than 30 per cent weight) and abundant water soluble side chain (more than 70 per cent) is proposed as a siRNA carrier with prolonged blood circulation time.
The CCC with higher density of graft chains have shown stronger interactions with siRNA than the lower density ones, suggesting that highly dense brushes of water soluble side chains enhance interpolyelectrolyte complex between the polycationic backbone and siRNA. The brush-like side chains might decrease dielectric constant around the polycation backbone. Such microenvironment of lower dielectric constant has been suggested to be responsible for enhancing ionic and hydrogen bonding interactions between the polycationic backbone and siRNA.
The siRNA complexed with the CCC was reported to be resistant to nuclease in 90 per cent plasma for 24 hours in vitro. Even when the CCC and siRNA were separately injected into mouse at 20 minutes interval, blood circulation of post-injected siRNA was found to be significantly increased. Studies demonstrated higher selectivity of CCC in its ionic interactions than other anionic substances in blood stream.
Various ionic components such as lipids, proteins and polysaccharides in plasma destabilise polyplexes between cationic copolymers and siRNA. The cationic polymers are rapidly eliminated from blood circulation due to their strong and non-specific uptake by the reticuloendothelial system (RES); the dense brushes shield the positive charges of cationic polymer and reduce its non-specific interactions with blood components, suppressing clearance of CCC by RES.
Hyaluronic acid (HA) nanogels: For target-specific intracellular delivery of siRNA to HA receptor over-expressing cancer cells, nano-sized HA hydrogels, called HA nanogels have been developed.
HA nanogels cross linked with disulfide linkages are prepared by an inverse emulsion method under ultrasonication conditions. The mean diameter of these nanogels is about 200nm. siRNA is encapsulated within the nanogels without any structural damage. HA nanogels are transported into the cells mainly by CD44 receptor mediated endocytosis. Release of siRNA from HA nanogels is achieved by glutathione. Glutathion (GSH) is a major intracellular reducing agent which is abundantly present in the cytoplasm. HA nanogels are stable in an extracellular condition but in the reducing environment of the cytoplasm, the nanogels are degraded and disintegrated by cleavage of the -S-S- linkage upon exposure to GSH releasing the entrapped siRNA in the cytoplasm.
Dynamic polyconjugates (DPC) for in vivo siRNA delivery: It has recently been demonstrated that the masked endosomolytic approach can enable highly efficient and functional delivery in vivo for siRNA.
These siRNA delivery vehicles have been assembled by first conjugating the siRNA to the synthetic, endosomolytic poly butyl and amino vinyl ether (PBAVE) via a labile disulfide bond. The subsequent modification of the amines of the PBAVE polymer with new maleamate derivatives containing PEG or galactose moieties, offered both shielding of the PBAVE's endosomolytic potential and hepatocyte-targeting. It has been observed that after entering the endosomes, the bond between the endosomolytic agent and masking agent is irreversibly broken and the masking compound becomes separated from the endosomolytic agent, thereby unleashing the masked endosomolytic agent's innate endosomolytic action. The endosome is disrupted and the biologically active compound is free to enter the cytoplasm.
The modular design of the DPC system has been shown to facilitate the incorporation of other polymers, labile linkages and targeting ligands to not only improve the system but also enable targeting other cells. Small size of the DPCs (<20 nm), suggests the possibility of tumour targeting.
A multifunctional & reversibly polymerisable carrier for efficient siRNA delivery: It is believed that a vehicle containing properties like pH-sensitive amphiphiliciy and environmentally sensitive siRNA release will be very efficient in siRNA delivery. Novel polymerisable surfactants include all these desired features. It contains polymerisable surfactants containing dithiol groups, protonable amines and lipophilic units. Different polyamines, including ethylenediamine, triethylenetetraamine and pentaethylenehexamine can be used to build the protonable head groups. Fatty acids of different chain length and structures, including lauric acid, stearic acid and unsaturated oleic acid can be used as the hydrophobic units.
The protonable amino groups have been designed to complex with siRNA via charge-charge complexation to produce compact nanoparticles (160-260 nm). Hydrophobic units are introduced to promote the formation of nanoparticle via hydrophobic condensation. The dithiol groups can be polymerised by forming disulfide bonds via autooxidation to further stabilise the nanoparticles.
Numerous amphiphilic materials are able to disrupt cell membranes. The pH-sensitive amphiphilicity of this siRNA carrier will allow the carrier to change its amphiphilic structure at endosomal-lysosomal pH (5.0-6.0), resulting in disruption of endosomal-lysosomal membranes and escape of siRNA delivery systems. It is important for these delivery systems to have low amphiphilicity at physiological pH, in order to minimise non- specific cell membrane disruption and high amphiphilicity at the endosomal pH, which will allow selective endosomal-lysosomal membrane disruption. The protonation may be governed by the overall pKa of the head groups of the surfactants, thus the pH-sensitive amphihilicity of the carriers can be tuned by modifying the structures of the protonable amines. After releasing from the endosome as the nanoparticles enter the cytoplasm, the disulfide bonds get reduced in the cytosol by glutathion, facilitating the dissociation of the nanoparticles and the release of siRNA.
Methods of improving conventional delivery vehicles
Photochemical internalisation (PCI): This technology employs specific photosensitising compounds which accumulate in the membranes of the endocytic vesicles. It has been observed that upon illumination, such photonsensitisers (For e.g. TPPS2a (meso-tetraphenylporphine with two sulfonate groups) become excited, and subsequently induce the formation of reactive oxygen species, primarily singlet oxygen. The highly reactive intermediate can damage cellular components, but the short range of action and short life time, confine the damaging effect to the production site. This localised effect induces the disruption of the endocytic vesicles, thereby releasing the entrapped siRNA into the cytosol. One of the major advantages of PCI as a delivery tool is its intracellular site-specific action. The fact that the photochemical-induced delivery of the specific macromolecule is confined only to the illuminated area is an advantage for in vivo studies. siRNA delivery would be limited to the desired cells, thereby further reducing non-specific effects. Increasing photochemical doses can, however, be associated with a higher degree of cytotoxicity.
RNAi gene silencing using cerasome as a viral-size siRNA-carrier free from fusion & cross-linking: Trialkoxysilylated quaternary ammonium forms single-walled liposomes to give surface-rigidified cerasomes, ceramic- or silica-coated liposomes.
It has bee observed that the surface-rigidified, infusible cerasomes (d = 60-70 nm) are not cross-linked when interacting with short siRNAs (7 nm), thus keeping the cerasome-siRNA complexes in a compact viral-size for their better RNAi performance, since size control of particles is crucial for their efficient cellular uptake.
The normal liposomes formed by non-ceramic lipid undergo fusion upon interaction with siRNA and therefore may not be taken up by the cells as the size increases in the process.
The size stability and serum-compatibility of the complexes suggest a potential in vivo application of the cerasome-mediated siRNA delivery.
Use of quantum dots: Quantum dots (QDs) are bright, photostable CdSe/ZnS fluorescent nanocrystals that exhibit tunable emission properties for a wide range of colour possibilities. Co-transfection of siRNA with QDs using standard transfection is found to be helpful in tracking delivery of nucleic acid, sorting cells by degree of transfection as cellular fluorescence correlate with level of silencing. Compared to alternative RNAi tracking methods, QDs exhibit superior photostability. Combining QDs with siRNA for RNAi tracking does not require chemical labelling of siRNA, which is costly and can potentially deter complexing with RNA-induced silencing complex.
Thus, QDs are versatile, photostable probes that offer an added dimension to improve the power of RNAi as an experimental tool.
Magnetic nanoparticles: These are made of iron oxide, which is fully biodegradable, coated with specific cationic molecules. Further these vehicles are attached with three dye molecules. These are further attached to an average of four membrane-penetrating molecules known as myristoylated polyarginine peptide (MPAP) to the nanoparticle surface. Finally, an average of five siRNA molecules is linked to their multifunctional core.
Nanoparticles' association with the siRNA is achieved by salt-induced colloidal aggregation and electrostatic interaction. It is possible to track the path of the nanoparticles using both magnetic resonance imaging, which detects the magnetic iron oxide core and fluorescence optical imaging, which detects the fluorescent molecules attached to the nanoparticle surface. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. In this way, the magnetic force allows a very rapid concentration of almost the entire quantity of the applied vector dose onto cells. The transfection rate can thus be clearly increased - also with difficult to transfect cells such as primary cells and the amount of nucleic acid required for successful transfection can be significantly reduced which in turn further reduces cytotoxicity. The cellular uptake of the genetic material is accomplished by endocytosis and pinocytosis.
The nucleic acids are then released into the cytoplasm by either the proton sponge effect caused by cationic polymers coated or the destabilisation of endosome by cationic lipids coated on the particles that release the nucleic acid into cells by flip-flop of cellular negative lipids and charge neutralisation.
The magnetic nanoparticles do not influence cellular functions. Also, the biodegradable cationic magnetic nanoparticles are not toxic at the recommended doses and even higher doses.
Multifuctional envelope-type nano device (MEND) :
The MEND consists of a condensed core and a lipid envelope structure equipped with the various functional devices. SiRNA are condensed into a compact core prior to inclusion into a lipid envelope. The first step of condensation allows protection, size control and improvement in packaging efficiency. In the second step, complexes are incorporated into lipid envelope.
The MEND is constructed by a novel assembly method called the lipid film hydration method. This method is based on packaging in three consecutive steps. They are:
● siRNA condensation with polycations
● Hydration of the lipid film for electrostatic binding of the condensed siRNA
● Sonication to package the condensed siRNA with lipids
This packaging mechanism is based on electrostatic interactions between siRNA, polycations and lipids.
The R8 peptide has been investigated as a functional device of the MEND because of its possibility to enhance cellular association and induce efficient cellular uptake via nonclassical endocytosis, which can circumvent lysosomal degradation. STR-R8 has been found to be able to compact siRNA to form nano-size particles (<100 nm) where the hydrophobic stearyl moiety is said to be critical in aiding compaction of siRNA into small particles. siRNA-MEND can thus deliver compact siRNA nanoparticles into cells to produce an efficient and persistent silencing effect with minimum cytotoxicity.
Thermally sensitive cationic polymer nanocapsules: Another way of enhancing gene silencing by improving endosomal escape is thermally sensitive cationic polymer nanocapsules (NC). Temperature-sensitive Pluronic/PEI2K nanocapsules have been synthesised by interfacial crosslinking reaction between pre-activated pluronic and low MW PEI (Mw. 2000, PEI2K) at an oil in-water interface during a modified emulsification/solvent evaporation process. The siRNA is linked to PEG via disulfide linkage and it interacts with cationic condensing agents to form core/shell-type complex micelles having a size of ~100 nm.
Stable siRNAPEG/NCs complexes with a size of ~100 nm at 37 °C are first taken up by the cells by an endocytosis process. Immediately after the enodocytosis, they are entrapped within the endosomal compartments having an average vesicle size of ~200 nm. After a brief cold shock at 15°C, siRNA-PEG/NCs complexes undergo a volume transition from a collapsed state (~100 nm) to a swollen state (~400 nm). An abrupt volume expansion of NCs beyond the critical size of an endosome vesicle physically disrupts the endosomal compartment, thereby releasing siRNA-PEG/NCs complexes into the cytosplasmic region. The escaped siRNA-PEG conjugate is decomplexed from the NCs by unknown mechanisms (no clear evidence for the decomplexation of DNA from polyplexes was proposed so far). An intact siRNA part is subsequently cleaved from the PEG part by abundant gluthathione species that maintain a reductive cytoplasmic microenvironment.
Conclusion
Although much research has been done in various laboratories, more studies related to the properties of these delivery vehicles need to be executed for the vehicles to be successful in the clinical trials and their final approvals in the markets. Further studies in the areas of toxicogenomics and biodistribution of siRNA will enable refining the delivery strategies for improved targeting for any given clinical condition. It is also envisaged that ultimately the development of safer siRNA targeted delivery systems that are biocompatible and genocompatible (in that they avoid immune stimulation and off-target gene effects), will lead to an expansion in the clinical roles of this technology.
(The authors are with Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Mumbai)