RNA interference technology with emphasis on delivery vehicles—prospects and limitations

Abstract

Context: RNA interference (RNAi)-based therapeutics rely upon safe and efficient delivery of small interfering RNA (siRNA) molecules. Objective: This review explores various dimensions of RNAi with emphasis on the development of nanoparticle-based delivery vectors for safe and efficient siRNA delivery. Methods: An exhaustive database search has been done regarding studies done to investigate the potential of siRNA delivery employing nanoparticles has been cited in the present review. Results and conclusion: With the current challenges, there is a need for collaborative work allowing for the successful development of nanoparticle/siRNA complexes as health-promoting biotherapeutics.

Keywords::

• gene silencing
• nanoparticles
• RNA interference
• small interfering RNA
• transfection

Introduction

Since the discovery of RNA interference (RNAi), a tremendous amount of effort has been directed toward the use of this technology for the development of therapeutics. A plethora of studies have appeared citing applications of RNAi in studies defining gene silencing and gene function using mammalian cells. RNAi is a highly conserved process of selective post-transcriptional down-regulation of target-specific genes in the cells. RNAi is mediated by small double-stranded RNA (dsRNA) sequences of 21–23 nucleotides, known as small interfering RNA (siRNA). These siRNAs, in conjunction with multi-protein complexes, exhibit the potential to degrade the gene-coding messenger RNA (mRNA) molecules which are complementary to one of the siRNA strands. dsRNA-mediated gene silencing was reported for the first time by Fire et al. in Caenorhabditis elegans (Fire et al. 1998). Since then, RNAi has evolved as a gold standard in studies related to the elucidation of gene function in basic biological research.

Though small-molecule inhibitors and monoclonal antibodies have achieved considerable success in therapies for deadly diseases such as cancer, they are based on the concept of traditional drug design, where the therapeutic intervention occurs at the protein level (i.e., after a disease-causing gene has already been translated into protein) (Druker et al. 2006, Smith et al. 2007). These traditional drugs were discovered through cumbersome processes of high-throughput screening followed by hit and trial in chemical modifications for lead compounds. Although several important and revolutionizing therapeutics have been successfully developed using the traditional approaches, there remain additional challenging diseases where the success of traditional drugs remains inefficient. However, RNAi based-therapeutics, by regulating the post-transcriptional levels of a disease-causing protein in a highly sequence-specific manner, should outshine traditional drug design. Moreover, RNAi-based therapeutics can allow for the exploration of new avenues of protein-targeting, which appears intractable and time-consuming with traditional drug design strategies.

Although RNAi holds great potential, its therapeutic applications are hindered by poor cellular uptake of siRNA molecules and their rapid enzymatic degradation (Zhang et al. 2007). Owing to their large molecular weight (∼13 kDa) and strong anionic charge due to the presence of a phosphodiester backbone (∼40 negative phosphate charges), naked siRNA is incapable of freely crossing the cell membrane. The electrostatic repulsion from the anionic cell membrane surface results in the failure of siRNA to passively diffuse through the cell membrane appears to be one of the major barriers to RNAi success. In diseases such as cancer, it is highly desirable that siRNAs reach specific diseased organs via systemic delivery routes. Moreover, naked siRNA has a very short half-life of < 1 h in human plasma; the administered siRNAs are rapidly cleared by the reticuloendothelial system (RES) composed of macrophages and Kupffer cells (Seow and Wood 2009). Unfortunately, for successful RNAi, the siRNA must reach the cytoplasm of the target cells. Hence, there arises an urgent need for a delivery vector to administer siRNA safely, effectively, and repeatedly to target tissues. The vector is expected to provide protection against enzymatic degradation, possess target specificity, enhance intracellular uptake, and escape from the endosome or lysosome into the cytosol, all resulting in efficient gene knockdown. Viral vectors originally emerged as a choice for siRNA delivery. However, safety issues related to oncogenicity, inflammation, and immunogenicity pose serious challenges toward their clinical application (Xu et al. 2005, Zaiss and Muruve 2005). The search for safer alternatives led to nanotechnology-based delivery vectors such as liposomes, nanotubes, and nanoparticles for efficient and target-specific siRNA delivery.

Nanoparticles are sub-microscopic colloidal particles in the nanometer-size range, with at least one dimension ≤ 100 nm that can entrap or adsorb drug molecules, proteins, or nucleic acids (Templeton and Lasic 1999). The small size facilitates nanoparticle movement in the thin capillary beds of tissues, thereby leading to delivery of the entrapped therapeutic molecule to the different tissues of the body (Gaumet et al. 2008). Moreover, nanoparticles can be easily engulfed by the cells, and thus can be used to deliver drugs to intracellular targets such as the cytoplasm, mitochondria, or nucleus (Panyam and Labhasetwar 2003). This review explores various dimensions related to the therapeutic potential of RNAi, that is, the mechanism of action, existing challenges, and future perspectives. Focus will be given toward nanotechnology-based approaches for the delivery of exogenous, synthetic siRNA employed for in vitro and in vivo studies. The final part of the article will revolve around the vision and expert opinion for the development of RNAi as a therapeutic entity.

Mechanism of RNA interference

The mechanism of RNAi mediated via siRNA takes place in the cytoplasm of the cells (Figure 1). RNAi is a well-orchestrated multi-step process which is initiated with the binding of dsRNA molecules to a specific endonuclease (a cytoplasmic ribonuclease III (RNase III)-like protein) called Dicer, which is capable of cleaving long dsRNAs (Bernstein et al. 2001). The conjugation of Dicer is followed by cleavage of long dsRNA into smaller duplexes with 19 paired nucleotides and two nucleotide overhangs at both 3′-ends; these small dsRNAs hence generated are called siRNAs (Elbashir et al. 2001). The generated siRNA molecules further bind to a nuclease-containing multi-protein complex called RNA-induced silencing complex (RISC) (Hammond et al. 2000). The RISC complex possesses RNA helicase activity which facilitates unwinding of the double-stranded siRNA molecule and removal of the sense strand, which is further degraded by cellular nucleases (Nykanen et al. 2001). The antisense strand of siRNA remains engaged with the RISC complex and directed toward the target mRNA sequence, where it anneals complementarily by Watson–Crick base pairing. In the following step, the RISC complex cleaves the target mRNA by endonucleolytic activity, thereby preventing its translation into protein (Martinez and Tuschl 2004). The endonucleolytic activity of the RISC complex is due to the presence of a cleavage enzyme called Argonaute 2 (AGO2) (Liu et al. 2004). Relative to the 5′ end of the siRNA guide strand, the targeted mRNA is cleaved between bases 10 and 11 (Elbashir et al. 2001). Finally, the cleaved products are released and degraded by cellular nucleases which liberate the RISC complex, and this allows further search for more target mRNAs to proceed with its interference activity (Leung and Whittaker 2005).

Figure 1. Mechanism of RNAi. siRNA is generated as Dicer cleaves dsRNA molecules. Incorporation of siRNAs into the RISC assembly, followed by unwinding of the double-stranded molecule due to helicase activity of RISC. Once the sense strand is removed, the antisense strand binds to targeted mRNA. RISC then cleaves mRNA that is then degraded by cellular nucleases.

Barriers to siRNA therapeutics

Delivery vectors play a crucial role in defining the success of siRNA-based gene silencing. The timely and efficient delivery of siRNA to the target site is the decisive factor for success. However, numerous challenges are being faced by siRNA during their delivery process, hampering their efficacy. Based on the mode of study, the barriers can be divided into two broad categories—in vitro and in vivo.

In vitro barriers

Though the site of action for siRNA is quite different from that of DNA, the former being functional in the cytoplasm and the latter in the nucleus, the barriers to be overcome by the two appear similar, from cell targeting to internalization and endosomal escape (Grayson et al. 2006, Katas and Alpar 2006). During the complexation process, siRNA is condensed due to electrostatic interaction between amino groups present in the polycationic polymers and phosphate groups of siRNA. Various factors like siRNA concentration, pH, buffer type, and charge ratio of polymer to DNA (N/P ratio), define the size of the complexes formed. The vector/siRNA complexes, in the 50–200 nm size range, mostly enter the cells via endocytosis or pinocytosis (Gao et al. 2005, Aoki et al. 2004). The first major obstacle that has to be overcome involves preventing the aggregation of complexes in the extracellular environment and obtaining a size that can be internalized. Incorporation of poly (ethylene glycol) (PEG) or sugar molecules, for example, cyclodextrin and hyaluronic acid (HA) at the surface of nanoparticles, is performed to avoid aggregation (Bartlett et al. 2007, Lee et al. 2007). The large size (∼13 kDa) and presence of polyanionic charges on siRNA hamper their efficient uptake by cells. In order to enhance the uptake of siRNA, the initial modifications reported the use of cell-penetrating peptides (CPPs) and cholesterol (Moschos et al. 2007, Muratovska and Eccles 2004). Various polycationic formulations comprising chitosan, polyarginine, polylysine, histidylated polylysine peptides, polyethylenimine (PEI), and polyamidoamine dendrimers have been used to deliver siRNA (Malhotra et al. 2009, Malhotra et al. 2011, Malhotra et al. 2013a, Malhotra et al. 2013b, Malhotra et al. 2013c).

The complexes, once internalized, become fused complexes bearing intracellular vesicles with the endocytic vesicles. The complexes escape endosomal degradation, for which various hypotheses have been proposed. The most accepted hypothesis of endosomal escape involves the presence of a cationic polymer, for example, polyamidoamine dendrimers or polylysine, leading to the physical disruption of the negatively charged endosomal membrane because of its direct interaction with the cationic polymer (Zhang and Smith 2000). Other strategies exploit the usage of either reduction-sensitive or pH-responsive polymers. PEI has ionizable amino groups, and therefore, by proton sponge effect, favors the release of endocytosed polyplexes (Boussif et al. 1995, Thomas et al. 2005, Thomas and Klibanov 2002, Akinc et al. 2005). Kim et al. reported that the polyaspartamide derivative (poly(PAsp(DET)) undergoes efficient endosomal escape because of a pH-dependent protonation of N-(2-aminoethyl)-2-aminoethyl groups in the PAsp(DET) side chain (Kim et al. 2010). Block copolymers of dimethylaminoethyl methacrylate (DMAEMA) and propylacrylic acid (PAA), which undergo structural rearrangements due to the acidic pH of endosomes, are also being investigated for siRNA delivery (Convertine et al. 2009). The block consists of PAA and a cationic complexation component which mediates a hydrophilic-to-hydrophobic transition at the endosomal pH, causing disruption of the membrane. Thermosensitive cationic polymeric nanocapsules are also employed to deliver siRNA. These nanocapsules can cause physical disruption of the endosome, due to temperature-induced swelling (∼119 nm at 37°C and ∼412 nm at 15°C) (Lee et al. 2008).

The stability of nanoparticles is highly desirable for extracellular siRNA protection, but at the same time, in order to mediate gene silencing, it is required to decomplex and release siRNA. Hence, for efficient silencing by siRNA complexed with nanoparticles, a subtle balance between protection and release of siRNA should exist (Zhang et al. 2009, Zhang et al. 2010). To facilitate this process, bioresponsive nanoparticles have been used, where release of siRNA occurs in response to an intracellular stimulus. For example, incorporation of acid-labile ketal linkages to PEI has been found to enhance transfection and RNAi. Ketalized PEI/siRNA polyplexes resulted in higher gene silencing efficiency than unmodified linear PEI (Shim and Kwon, 2009), because of selective cytoplasmic localization of the polyplexes and the efficient disassembly of siRNA from the polyplexes. Polyplexes containing reducible polycations are also being studied for their capability as siRNA delivery vehicles. These polycations degrade in response to cytoplasmic redox conditions. Under physiological conditions, the reducible poly(amido ethylenimine) efficiently condenses siRNA to form stable complexes, and in presence of a reducing environment, results in complete release of siRNA (Hoon Jeong et al. 2007). A hundred-fold higher siRNA transfection efficacy was observed with the polyion complex (PIC) micelle prepared with a disulfide crosslinked core through the assembly of iminothiolane-modified poly(ethylene glycol)-block-poly(L-lysine) [PEG-b-(PLL-IM)] and siRNA, in comparison to non-crosslinked PICs (Matsumoto et al. 2009). On one hand, siRNA is well protected from degradation and non-specific clearance by PICs in the extracellular milieu, and on the other hand, owing to the selective release of siRNA in the intracellular milieu, higher transfection efficiency is observed. Also, siRNA grafted to poly(aspartic acid) [PAsp(-SS-siRNA)] via a disulfide linkage was found to give a higher siRNA efficiency due to efficient siRNA release from the PIC under intracellular reductive conditions (Takemoto et al. 2010).

In vivo barriers

The route of siRNA administration, whether intravenous, intranasal, intratracheal, subcutaneous, intratumoral, intramuscular, or oral, depending on the targeted disease, defines the in vivo barriers of siRNA therapeutics. Degradation by nuclease activity in plasma is one of the most significant biological barriers encountered by systemically administered siRNA. The major degradative enzymatic activity that occurs in the plasma is that of 3′ exonuclease; although on a smaller scale, cleavage of internucleotide bonds can also take place. Thus, one major challenge is to protect siRNA in serum. For this purpose, several chemical modifications have been proposed, including modifications of the sugars or the backbone of siRNA by 2′-O-methyl and 2′-deoxy-2′-fluoro (OMe/F) or phosphorothioate linkages (Akhtar and Benter 2007). However, polymeric nanoparticle-complexed siRNA can easily bypass this barrier as it has no available sites for 3′ nuclease binding and cleavage. Efficient siRNA delivery faces another major problem in the form of rapid clearance by the RES. Endocytosed siRNA, as well as their carriers, are engulfed by Kupffer cells in the liver and spleen macrophages (Alexis et al. 2008). Opsonins, which are composed of immunoglobulins, complement system proteins, and other serum proteins recognize the nanoparticles used to deliver siRNA as foreign particles in the body. Once opsonized, these particles are then taken up by a variety of receptors present on the cell surface of macrophages. Immunoglobulin G-opsonized particles are recognized by Fc receptors and complement-opsonized particles are internalized through complement receptors, leading to their degradation. RES-associated clearance can be overcome by the modifications of nanoparticle surfaces with hydrophilic polymers such as PEG that reduce the adsorption of opsonins and clearance by phagocytosis (van Vlerken et al. 2007). One of the most crucial steps involved in efficient gene silencing via siRNA is the specific recognition and binding to the target mRNA in the cytosol. Nanoparticle-complexed siRNA delivery is not free of adverse effects, and some of the off-target effects observed include toxicity, stimulation of immune response, inflammation, and undesirable effects on other genes (Aigner 2006). Chemical modifications of siRNA where uridine bases are replaced by their 2′-fluoro-, 2′-deoxy-, or 2′-O-methyl-modified counterparts have been reported to block immune recognition of siRNAs by toll-like Receptors (TLR) (Sioud 2006). Hence, another strategy has been proposed that involves the use of polymers which avoid the delivery into the endosomes or block TLR signaling to deliver siRNA. In principle, various factors should be taken into consideration both at the cellular and at the whole-organism level to design and develop efficient siRNA delivery vectors.

Nanoparticle-mediated siRNA delivery

Several polymers like PEI, chitosan, and PEG have been used to develop nanoparticles that can be further employed to deliver siRNA. In order to mediate targeted delivery via receptor-mediated endocytosis, surface ligands are used to modify the polymers that possess derivable functional groups. Chitosan is an aminoglucopyran that possesses randomly distributed N-acetylglucosamine and glucosamine residues. Chitosan has emerged as a promising candidate due to its versatile biological activity, biocompatibility, and biodegradability, along with low toxicity (Rinaudo 2006). Chitosan has largely become an efficient vector, and its efficacy in the delivery of siRNA is governed by several structural and experimental parameters such as molecular weight (MW) and degree of deacetylation (DDA). Katas et al. seems to be the first group to have used chitosan in vitro as a delivery vehicle for siRNA (Katas and Alpar, 2006). To investigate the gene-silencing efficiency of chitosan nanoparticles, two different types of cell lines, namely, Chinese hamster ovary cells (CHO K1) and human embryonic kidney cells (HEK 293) were used (Table I). The study revealed that the silencing effect of siRNA was in direct correlation with the method of association of chitosan with siRNA. When compared, it was observed that nanoparticles of chitosan/tripolyphosphate (TPP)-entrapping siRNA were better vectors for siRNA delivery than simply chitosan, possibly due to their high binding capacity and loading efficiency. Another group, Howard et al. engineered a novel chitosan-based siRNA nanoparticle delivery system for in vitro and in vivo RNAi. The study showed nanoparticle-mediated knockdown of endogenous Enhanced Green Fluorescent Protein (EGFP) in both human lung carcinoma cells (H1299) and murine peritoneal macrophages, with 77.9% and 89.3% fluorescence reduction, respectively (Howard et al. 2006). When chitosan/siRNA formulations where nasally administered in bronchiole epithelial cells of transgenic EGFP mice, efficient in vivo RNAi was achieved (37% and 43% reduction compared to mismatch and untreated control, respectively) (Howard et al. 2006). These findings highlight the novel chitosan-based system for its potential therapeutic application in RNAi-mediated therapy of systemic and mucosal disease.

Table I. Nanoparticles employed to deliver siRNA.

Vector	Proposed study	Outcome	Reference
Chitosan/TPP nanoparticles	In vitro gene silencing in CHOK1 and HEK293 cells	Chitosan/TPP nanoparticles are better vectors as compared to chitosan only.	Katas and Alpar, 2006
Chitosan/siRNA complexes	EGFP gene-silencing in human lung cancer cell line H1299 & murine macrophage	Development of an efficient siRNA nanocarrier that can be used for both in vitro and in vivo RNA interference protocols	Howard et al. 2006
Polyguluronate-coated chitosan/siRNA nanoparticles	Gene-silencing in HEK 293 T and HeLa cells	Efficient siRNA delivery	Lee et al. 2009
Chitosan/siRNA nanoparticles	FHL2 gene-silencing in SW40 & HeLa cells	FHL2 gene expression silenced by 69.6%	Ji et al. 2009
PEI/siRNA complexes	Subcutaneous administration of complexes to downregulate HER-2 expression in mice tumor models	siRNA downregulation & marked reduction in tumor growth	Urban-Klein et al. 2005
PEGylated PEI-RGD/siRNA self-assembling nanoparticles	siRNA delivery against VEGF R2 and tumor angiogenesis	Sequence-specific reduction of protein expression by siRNA	Schiffelers et al. 2004
PEI-alginate/siRNA nanoparticles	Deliver siRNA into COS-1 cells	80% suppression of GFP expression	Patnaik et al. 2006
Acylated PEI-PEG nanoparticles	Deliver siRNA into HEK 293 cells	85% inhibition of GFP expression	Nimesh and Chandra 2009
PEI nanoparticles to deliver siRNA against VEGF	Deliver siRNA against VEGF	Knockdown VEGF in pancreatic and prostate cancer models	Höbel et al. 2010
Cyclodextrin-containing polymers (CDP)	siRNA delivery targeted against the EWS-FLI1 gene	Significant inhibition of tumor growth	Hu-Lieskovan et al. 2005
Transferrin-targeted CDP nanoparticles	siRNA delivery to tumor cells	50% reduction of tumor luciferase activity	Bartlett et al. 2007
siRNA/CDP nanoparticles	Administration of siRNA to patients with solid cancers	Intracellular localization in melanoma patients	Davis et al. 2010
PEI modified with HA	siRNA delivery to B16F1, A549, HeLa, and Hep3B tumor cells	Survivin-specific siRNA resulted in significant reduction in the expression of mRNA	Han et al. 2009
Biodegradable poly(amido amine)s	siRNA against epidermal growth factor receptor (EGFR)	80% gene knockdown with low cytotoxicity	Vader et al. 2011
Biodegradable pHPMA-MPPM and TMC polymers	siRNA delivery to In H1299 cells	30–40% reduction in luciferase gene expression	Varkouhi et al. 2011
Triblock copolymer, of monomethoxy poly(ethylene glycol), poly(e-caprolactone) and poly(2-aminoethyl ethylene phosphate)	Delivery of siRNA to BT474 breast cancer cells	Efficient inhibition of tumor growth in a BT474 xenograft murine model	Mao et al. 2011

Using a coacervation method, size-controlled chitosan nanoparticles were prepared in the presence of polyguluronate (PG)-encapsulating siRNA (Lee et al. 2009). The siRNA-loaded chitosan nanoparticles formed were found to possess a mean diameter in the range of 110–430 nm, depending on the weight ratio between chitosan and siRNA. These nanoparticles efficiently delivered siRNA to the cell lines, HEK 293 T and HeLa, with reduced cytotoxicity (Table I). In another study done on chitosan/siRNA nanoparticles, it was observed that these complexes possess irregular lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and a net positive charge, with zeta potential value of 58.5 mV (Ji et al. 2009). Gene knockdown studies done with chitosan/siRNA nanoparticles showed that chitosan/siRNA nanoparticles silence FHL2 gene expression by 69.6% (FHL2 is an oncogene highly expressed in various types of cancer cells, such as epithelial ovarian cancer, hepatoblastoma, colon cancer cell line—SW480, cervical cancer cell line—HeLa, and some breast cancer cells). It was also observed that in the human colon adenocarcinoma LoVo cells, siRNA-mediated reduction in FHL2 expression inhibited the growth and proliferation of human colorectal cancer cells. These results, therefore, suggest chitosan/siRNA nanoparticles as being efficient in vitro delivery systems.

PEI, designated as the gold standard of gene transfection, has high cation density (a positive charge per 43 Da, the monomer's MW), and is one of the most widely explored cationic polymers for gene delivery. Since the first successful application by Behr et al. of PEI-mediated oligonucleotide delivery, it has been further derivatized to improve the physicochemical and biological properties of polyplexes (Boussif et al. 1995, Neu et al. 2005). It is used for the formation of highly condensed particles by interacting with nucleic acids. PEI/siRNA complexes are more stable and uniform-sized particles as compared to DNA (Grzelinski et al. 2006). The interaction of PEI and siRNA is non-covalent in nature. The complexation of synthetic siRNA with low MW PEI efficiently stabilizes siRNAs and delivers it into cells, with no toxicity and complete bioactivity (Urban-Klein et al. 2005). Moreover, subcutaneous administration of these complexes in a mouse tumor model showed the delivery of intact siRNAs into the tumors. siRNA downregulated HER-2 expression, and a marked reduction in tumor growth was observed with PEI-complexed siRNAs targeting the c-erbB2/neu (HER-2) receptor that was injected intraperitoneally. Various targeting ligands have been attached to PEI to achieve targeted delivery. PEI that is PEGylated with PEG having an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end allows complexation with siRNA as self-assembling nanoparticles (Table I). These self-assembled nanoparticles then act as a means to target tumor neovasculature-expressing integrins and deliver siRNA that inhibits expression of vascular endothelial growth factor receptor-2 (VEGF R2) and tumor angiogenesis (Schiffelers et al. 2004). Intravenous administration of complexes in tumor-bearing mice showed selective uptake of siRNA at the tumor site, sequence-specific reduction of protein expression by siRNA, and inhibition of both tumor angiogenesis and growth rate. In one of our studies, we prepared an ionic complex of PEI (750 kDa) and alginic acid to amalgamate the properties of cationic PEI with the polysaccharide alginate (Patnaik et al. 2006). The PEI/alginate (6.26% amino groups derivatized by alginate) nanoparticles efficiently delivered siRNA into COS-1 cells, and about 80% suppression of GFP expression was observed. In another published siRNA delivery study, we prepared nanoparticles of PEI by acylating PEI with propionic anhydride followed by crosslinking with polyethylene glycol-bis(phosphate) (Table I). When siRNA nanoparticles were delivered into HEK 293 cells, up to 85% inhibition of GFP expression was shown, comparable to that for siRNA/Lipofectin transfection (81% inhibition) (Nimesh and Chandra 2009). Hobel et al. used PEI nanoparticles to deliver siRNA against VEGF, and they reported the success of these nanoparticles in effective knockdown of VEGF in pancreatic and prostate cancer models (Höbel et al. 2010). Further, they derivatized PEIs with maltose, maltotriose, or maltoheptaose (OM/PEI) to deliver siRNA in vitro and in vivo (Hobel et al. 2011). On systemic application in vivo, the OM/PEI/siRNA complexes show marked differences in the siRNA biodistribution profile, that is, substantially decreased siRNA levels in the liver and increased siRNA levels in the muscle.

Recently, cyclodextrin (CD) and CD-containing polymers (CDP) have been employed for siRNA delivery (Pun et al. 2004). In a study conducted in a murine model of metastatic Ewing's sarcoma, CDP-mediated siRNA delivery targeted against the EWS-FLI1 gene significantly inhibited tumor growth (Table I) (Hu-Lieskovan et al. 2005). These results coincided with minimal adverse effects, and long term tail vein administrations of these conjugates showed no abnormalities in interleukin-12 and interferon (IFN)-α, liver and kidney function tests, complete blood counts, or pathology of major organs. Positron emitting tomography (PET) demonstrated that both non-targeted and transferrin (TF)-targeted siRNA nanoparticles exhibited a similar biodistribution and tumor localization. siRNA nanoparticles targeting TF had tumor luciferase activity reduced by approximately 50% compared to non-targeted siRNA nanoparticles, 1 day after injection (Bartlett et al. 2007). Bartlett et al. conducted a study where siRNA/CDP nanoparticles were administered intravenously in A/J mice bearing subcutaneous Neuro2A tumors. Following three consecutive daily doses of TF-targeted nanoparticles carrying 2.5 mg/kg of two different siRNA sequences targeting RRM2, inhibition in tumor growth was observed (Bartlett and Davis 2008). When tested in non-human primates, it was found that TF-targeted siRNA/CDP nanoparticles were well-tolerated in cynomolgus monkeys at doses of 3 and 9 mg siRNA/kg by intravenous administration (Heidel et al. 2007). Recently, the first in-human phase I clinical trial for siRNA/CDP has been reported. The study involved the systemic administration of siRNA to patients with solid cancers using targeted CDP nanoparticles. Nanoparticles were shown to be intracellularly localized in melanoma patients, in the results of tumor biopsies that were done for those patients (Davis et al. 2010). Additionally, in comparison to pre-dosing tissues, reduction in the levels of RRM2-specific mRNA and protein were observed.

Recently, biodegradable polymers have emerged as attractive candidates for the design of siRNA-loaded polyplexes. Han et al. modified biocompatible hyaluronic acid (HA) with the cationic polymer PEI and demonstrated increased cellular uptake of siRNA with cationic PEI derivatives of HA (HA-PEI) in B16F1, A549, HeLa, and Hep3B tumor cells, as compared to PEI alone (Table I). Further, HA-PEI used to deliver a survivin-specific siRNA resulted in significant reduction in the expression of mRNA of the target gene in all of the aforementioned cell lines (Han et al. 2009). A new series of biodegradable poly(amido amine)s with disulfide linkages in the backbone has been developed by Vader et al. (Vader et al. 2011), out of N,N′-cystaminebisacrylamide (CBA), 4-amino-1-butanol (ABOL) and ethylene diamine (EDA). They designed siRNA against epidermal growth factor receptor (EGFR) and delivered with these polymers containing 25% or 50% EDA in 14 C cells. The results showed more than 80% gene knockdown with low cytotoxicity. In another study, Varkouhi et al. investigated two biodegradable cationic polymers for siRNA delivery—pHPMA-MPPM (poly((2-hydroxypropyl) methacrylamide 1-methyl-2-piperidine methanol)) and TMC (O-methyl-free N,N,N-trimethylated chitosan) (Varkouhi et al. 2011). In H1299 human lung cancer cells, expression of the luciferase gene was reduced by 30–40% when transfected with polyplexes formulated from pHPMA-MPPM and TMC, comparable to the results obtained with Lipofectamine. Further, application of photochemical internalization (PCI), along with addition of diINF-7 peptide to the formulations, increased their silencing activity up to 70–80%. Recently, Mao et al. reported a triblock copolymer consisting of monomethoxy poly(ethylene glycol), poly(e-caprolactone), and poly(2-aminoethyl ethylene phosphate). These nanoparticles, in aqueous solution, are self-assembled into micellar nanoparticles (MNPs) with an average diameter of 60 nm and a zeta potential of 48 mV (Mao et al. 2011). Efficient gene knockdown effect was observed in BT474 breast cancer cells once the micelleplex formed by the interaction of MNPs and siRNA and was internalized by cells followed by siRNA release. Furthermore, siRNA targeting the acid ceramidase gene, when condensed as a micelleplex, induced significant apoptosis and reduced proliferation of cancer cells. An in vivo study showed promising results as systemic delivery of these micelleplex not only efficiently inhibited tumor growth in a BT474 xenograft murine model with suppressed acid ceramidase expression, but also showed no activation of the innate immune response.

Advantages and limitations of nanoparticles

Several advantages are attributed to the nanoparticles prepared from polymers, such as ease of manipulation with further scope to change the molecular weight (MW), geometry (linear and branched), stability, safety, low cost, and high flexibility regarding the size of the transgene delivered. Various targeting moieties such as RGD peptides or transferrin can be used to tag nanoparticles to achieve target-specific siRNA delivery (Schiffelers et al. 2004, Davis, 2009). Owing to their submicroscopic size (usually 10–200 nm), nanoparticles can readily penetrate the cells and interact with biomolecules on the surface or inside the cell. Also, the small size of nanoparticles allows their penetration to the tumor tissues, in depth and with a high level of specificity, thereby improving the targeted delivery of drug/gene (Cuenca et al. 2006). Limitations are still associated with polymeric nanoparticles, despite their advantages and successful implications in numerous studies. The destabilization and ultimate rupture of the cell membrane occurs due to strong electrostatic interactions between the polycationic polymers constituting the nanoparticles and plasma membrane proteins (Fischer et al. 2003). The polycationic polymers were found to have the highest toxicity followed by neutral and anionic ones in a comparative study done between polycationic, neutral, and polyanionic polymers (Jevprasesphant et al. 2003). In order to circumvent this problem, several strategies based on the reduction of surface charge by coating with HA or PEG have been investigated (Jiang et al. 2008, Duan et al. 2010). Branched PEIs have been observed to be more toxic and less suitable for transfection, particularly at higher N/P ratio, than the linear ones (Wightman et al. 2001). A major disadvantage attached to PEI is the fact that it is a non-biodegradable polymer, and hence its accumulation within the cells could cause interference with vital intracellular processes (Godbey et al. 1999, Godbey et al. 2001). PEI/DNA complexes with a higher ratio of cation/anion can activate the complement system, but the extent of activation is lowered as the PEI/DNA complexes approach neutrality (Ferrari et al. 1997, Plank et al. 1996). Chemical modifications where small MW PEIs are coupled to generate higher MW molecules using degradable bi-functional linkages have been found to reduce toxicity. Chitosan possesses minimal toxicity and is found to be an efficient in vitro and in vivo siRNA delivery vector capable of mediating gene silencing (Howard et al. 2006, Pille et al. 2006). However, conditions for transfection using chitosan nanoparticles must be well-elucidated before their clinical applications, as chitosan suffers from low transfection efficiency and optimum conditions for transfection are not clear. Moreover, non-specific stimulation of innate immune inflammatory responses such as type I IFN production associated with RNA duplex interaction with endosomal toll-like receptors (TLR) can be potentiated with nanoparticle/siRNA delivery into the endosome (Marques et al. 2006). However, TLR-7 interaction and associated non-specific effects can be disrupted to an extent when 2′-O-methyl nucleotides is incorporated into the siRNA duplex strand (Judge et al. 2006).

Conclusion

RNAi discovery has become a milestone in the area of novel therapeutic approach research. However, RNAi, specifically siRNA application, has its own limitations due to low cellular uptake, short half-life in vivo, rapid clearance, and quick enzymatic degradation. All these associated disadvantages have forced advancement leading to the development of novel nanoparticle/siRNA delivery systems. Several delivery systems have been designed so far, and each has its own associated advantages and disadvantages. Various modifications that have been introduced, such as usage of PEG— done to increase residence time, and the use of cell-targeting peptides with the delivery systems, have greatly contributed to enhancing the efficacy of these delivery systems. Still, despite the advancements in this field of research, the major challenges to combat in order to ensure efficiency include the fate of the nanomaterials in vivo, their long-term side effects on gene expression and toxicity, as well the conventional methods of ADME/Tox. The efficacy and biosafety of nanoparticle/siRNA formulations need to be well monitored, with properly designed pre-clinical and clinical studies. A review of the recent advances in research suggested the use of polymeric nanoparticles to deliver functional siRNA to the target cells. Several studies have been conducted with nanoparticles on various tumor models to deliver siRNA for antitumor treatments. Though these successful research studies on animal models for systemic delivery to non-human primates have been a much awaited boost for scientists in the field, a number of hurdles and concerns must be overcome before RNAi can be harnessed as a new therapeutic modality (Zimmermann et al. 2006). The nanoparticles must possess 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 clinical side effects. Several nano-based vectors have been used for in vivo siRNA delivery to a variety of tumor xenograft models, with a view that they possess minimal antigenicity and high transfection efficiency. The results have shown a promising future for the technology, as remarkable suppression of tumor growth via siRNA/vector complexes was observed. However, lack of understanding of the mechanism and off-target interference by the complexes have posed a major obstacle toward development of siRNA-based therapeutics. These issues have thus raised the cry for more exhaustive and detailed planning for successful implications of siRNA complexes with efficacy and for pharmacokinetics of siRNA/vector systems in clinical applications. In this era of developing genetic therapeutics, there is a call for collaborative work by academicians and industry groups in order to develop methods for preparation of more stable polymeric nanoparticle/siRNA complexes and robust analytical methods to characterize formulations during formation and storage.

Acknowledgements

This work was supported by Science & Engineering Research Board (SERB), Department of Science and Technology (SERC Fast Track Proposals for Young Scientists Scheme), and Government of India.

Declaration of interest

The authors report no declaration of interest. The authors alone are responsible for the content and writing of the paper.