Lipid-based Nanoparticles in the Systemic Delivery of siRNA

Cationic Lipid-based siRNA Delivery

Cationic liposomes are the most common lipid-based nanocarrier used for siRNA delivery. siRNA loading onto the cationic liposome is mainly attributed to the electrostatic interaction between the anionic siRNA and cationic lipids. The cationic lipids facilitate the particles' intracellular uptake and further endosomal escape. Some cationic liposomes have been widely used for siRNA delivery, such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)–siRNA, N-(1-[2,3-dioleyloxy]-propyl)-N,N,N-trimethylammonium chloride–siRNA and Lipofectamine® 2000 (Life Technologies, MD, USA)–siRNA complexes,^[38–41] which are simply formulated by cationic lipids and siRNAs. However, these complexes generally have a high electric charge density on their surface that readily induce nonspecific interactions with serum proteins and immunogenic response, resulting in their rapid removal from the blood stream.^[4,42,43]

Development of Advanced Cationic Lipid-based siRNA Delivery System

Stable Loading of siRNA. To stabilize the cationic lipid-based siRNA delivery system, many neutral lipids, such as cholesterol (Chol) or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE),^[44] have been added to the complex formulation. These neutral lipids not only stabilize the formation but also enhance the cellular uptake of particles, which will be discussed later (see the 'Improving the cellular uptake & enhancing siRNA release into the cytoplasm' section).

In comparison with surface loading, siRNA loading in the NP core should provide better delivery stability. To improve the siRNA core loading efficiency, many helper cationic polymers, such as protamine, were introduced to precondense siRNA into the core of a liposome. A good example is the liposome–polycation–DNA (LPD) NPs developed by Li et al. [45]. In this NP, protamine interacts with the nucleic acid to form a negatively charged compact core, cationic liposomes composed of DOTAP/Chol collapse onto the core via charge–charge interaction and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–PEG is then coated onto the outer surface of the particles. Thus, the siRNA delivery is supported and shielded by both the cationic lipid bilayer and PEG coating.^[45]

To improve siRNA loading efficacy, many efforts have been undertaken to optimize the key component of liposome cationic lipids. Santel et al. synthesized a series of β-L-arginyl-2, 3-L-diaminopropionic acid-N-palmityl-N-oleylamide trihydrochloride (AtuFECT) cationic lipids and validated that AtuFECT01 enables enhancement of the siRNA-binding ability when compared with commercial cationic lipids, such as DOTAP or N-(1-[2,3-dioleyloxy]-propyl)-N,N,N-trimethylammonium chloride [7]. The ability of the AtuFECT-formulated liposome to deliver siRNA (the resultant AtuFECT–liposome–siRNA complex is termed as AtuPLEX) that inhibits CD31 and Tie2 in the vasculature of mice has also been demonstrated.^[7] Later, the same group extended the application of the optimized AtuPLEX to treat advanced solid tumor by targeting PKN3 (termed Atu027) and achieved significant inhibition of tumor growth,^[46] lymph node metastasis^[46] and lung metastasis formation.^[47] Notably, a Phase I clinical trial of Atu027 for the treatment of advanced solid cancers has been completed, and a further clinical test using a combination of Atu027 and gemcitabine is currently ongoing.^[201,202]

The stable loading of siRNA depends not only on the formulation components but also on siRNA-integrating methods. Buyens et al. published a comprehensive review on this aspect, which covers variable methods for siRNA loading, such as simple mixing, direct hydration of a lipid film by a siRNA-contained solution and the ethanol dilution method.^[48] Among them, the simple mixing method usually gave the poorest siRNA encapsulation efficacy.^[48]

Furthermore, the stable systemic delivery of siRNA can be improved by chemical modifications of the siRNAs backbone, such as 2'-O-methyl and 2'-fluoro RNA. Chol conjugation to the siRNA sequence also enhanced siRNA loading efficacy on lipid-based NPs, and exhibited higher biologic activity compared with unmodified siRNA.^[49]

Improving siRNA Delivery Pharmacokinetics. To improve the blood stability and the in vivo pharmacokinetics properties, many other helper lipids were introduced into the siRNA delivery system, such as PEGylated lipid (PEG lipid).^[48] The PEGylation effect of prolonging the circulation time of many carriers in the blood has been reviewed by many groups,^[48,50,51] including, but not limited to, the influence of acyl chain length in PEG lipids and their molar percentage in the liposome composition.^[52] Sonoke et al. found that the PEG lipids with long acyl chains showed better siRNA delivery pharmacokinetics and efficiency when compared with those with the short or unsaturated chains.^[53] The percentage of PEG lipids in the siRNA delivery system should consider both blood circulation time and cell uptake efficiency. A higher percentage of PEG lipids usually gives a longer blood circulation time but weakens the cellular uptake and subsequent endosomal escape. Therefore, an optimal density of PEG lipids is required.^[48,51]

Yagi et al. developed a 'wrapsome' NP, comprised of a core of siRNA/cationic DOTAP that was fully enveloped by a neutral lipid bilayer containing egg phosphatidylcholine and PEG lipid in a weight ratio of 24:14.8. They found that the wrapsome could improve the stability and systemic circulation of siRNA, thus resulting in enhanced specific gene knockdown and significant anti-tumor activity in vivo.^[54]

To overcome the PEGylation-induced problem of cell uptake and endosomal escaping, Carmona et al. developed a pH-sensitive PEGylated liposome by postcoupling a PEG-2000 dialdehyde on the surface of particles composed of cationic cholesteryl polyamine–N¹-cholesteryloxycarbonyl-3, 7-diazanonane-1, 9-diamine, neutral lipids (DOPE) and Chol–PEG350 aminoxy lipid via oxime linkage. The oxime linkage of the NPs is stable at pH 7, but is prone to decomposition in an acidic environment (pH 5.5 and below), which induces the release of PEG coating from particles, thus granting acidic pH-triggered cell uptake and endosome release of nucleic acids. This formulation demonstrated effective RNAi delivery for on the control of hepatitis B virus (HBV) virus replication.^[55] Hatakeyama et al. developed another PEG-cleavable siRNA delivery system, termed a 'multifunctional envelope-type nanodevice' (MEND), composed of DOTAP, DOPE, Chol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–PEG and a PEG–peptide–DOPE (PPD). PPD has a peptide sequence-linked PEG and DOPE lipid moiety together, so that the PEG is removed when the peptide linker is cleaved by the target molecule, such as MMP. It has been demonstrated that PPD-formulated NPs accelerated both cellular uptake and endosomal escape in MMP-rich tumor environments, compared with a conventional PEG fomulation, resulting in potent silencing (~70%) activity in vivo with no observed hepatotoxicity and innate immune stimulation.^[56]

Notably, the PEGylation methods also influence the stability of siRNA delivery. For example, simply mixing siRNAs with PEGylated liposomes results in poor loading of siRNAs onto the outer surface of particles, causing the premature release of siRNA, whereas post-PEG-coating on the siRNA liposome could enable more stable siRNA delivery. This aspect has been discussed and reviewed in detail by Buyens et al..^[48] Although the beneficial PEGylation effect on siRNA delivery is well acknowledged, it has been found that repeated injection of PEGylated NPs could induce the loss of their long circulation characteristics owing to the production of the anti-PEG IgM.^[57] The phenomenon of accelerated blood clearance serves as a reminder of the limitation of PEG-coated NPs.

In addition, other challenges in the context of the pharmaceutical development of lipid-based siRNA therapeutics have been reviewed extensively by Gindy et al., including the development of a robust manufacturing process, the setting of appropriate product specifications and controls, development of strategies to assess and ensure product stability, and the evaluation of product comparability throughout development.^[58]

Improving Biocompatibility & Reducing Immunotoxicity. To reduce the immunotoxicity of siRNA delivery, some biogenic materials were included in the cationic lipid-based siRNA delivery system. For example, hyaluronic acid, a biogenic component distributed widely in the ECM, was introduced into LPD-NPs by Chono et al. to develop a liposome–protamine–hyaluronic acid (LPH)-NP.^[59] Hyaluronic acid provides LPH-NP multivalent charges to enhance the particle condensation while containing no immunostimulatory CpG motifs. This LPH-NP delivery of siRNA induced a 80% silence of luciferase activity in the metastatic B16F10 tumor in the lungs at a low, single, intravenous injection dose of 0.15 mg siRNA/kg,^[59] while not causing obvious immunotoxicity at a dose range of 0.15–1.2 mg siRNA/kg. Recently, they employed LPH-NP in the delivery of siRNA for silencing of CD47, a 'self-marker' that is usually overexpressed on the surface of cancer cells to enable them to escape from immunosurveillance, and achieved effective inhibition of melanoma tumor growth and lung metastasis.^[60]

To improve the biocompatibility of cationic lipid delivery systems, Yang et al. developed a lipid–polymer hybrid NP prepared by a single-step nanoprecipitation of a formulation of cationic lipids, BHEM–Chol and amphiphilic polymers. Such hybrid NPs exhibited excellent stability in serum and showed significant improvement on biocompatibility compared with the pure BHEM–Chol particles.^[26] A similar toxicity reduction effect has also been observed for other hybrid NPs with involvement of polyglutamate.^[61–63]

Improving the Cellular Uptake & Enhancing siRNA Release into the Cytoplasm. To enhance the cellular uptake and further endosomal escaping of siRNA, some neutral helper lipids, such as DOPE and 1,2-distearoyl-sn-glycero-3-phosphocholine were added to the cationic lipid-based siRNA delivery system. The role of such fusogenic lipids has been reviewed by Wasungu et al..^[44] DOPE profoundly affects the polymorphic features of the liposome–siRNA complex (termed lipoplexes) in that it promotes the transition from a lamellar to a hexagonal phase, thus inducing fusion and disruption of the membrane. The optimized 3-β-(N-[N',N'-dimethylaminoethane] carbamoyl) Chol/DOPE-based lipoplexes have been found to improve the transfection efficiency of the lipoplex.^[64] Khoury et al. prepared a NP using the cationic lipid RPR209120 in combination with DOPE. This NP enables effective systemic delivery of DNA, resulting in successful silencing of TNF-α in collagen-induced arthritis of mice, and complete cure of collagen-induced arthritis via weekly intravenous administration.^[65] Since then, this formulation for siRNA delivery to myeloid cells has been successfully applied.^[66,67]

By replacing the protamine/DNA core of the LPD NPs with a pH-sensitive calcium phosphate (CaP) core, Yang et al. recently developed a biodegradable lipid/calcium/phosphate NP (LCP-NP). CaP is biocompatible, biodegradable material and native to the body, as it is the principle mineral component of teeth and bones. It is hypothesized that the CaP core could be rapidly dissolved at acidic endosome pH, which will increase the osmotic pressure and cause NP disassembly, endosome swelling and, finally, siRNA release into the cytoplasm. The study showed that the LCP-NP releases more cargo to the cytoplasm compared with the LPD formulation, leading to a significant (~40-fold in vitro and ~fourfold in vivo) improvement in siRNA delivery. More recently, they coformulated three siRNAs against MDM2, c-myc and VEGF in a LCP-NP. Such a formulation caused simultaneous silencing of the three oncogenes in metastatic nodules, and resulted in significant inhibition of lung metastases (~70–80%) at a relatively low dose (0.36 mg/kg) without any observed toxicity.^[68]

Recently, to enhance endosomal escape, Harashima et al. synthesized a pH-sensitive cationic lipid, YSK05, which contains a tertiary amine group for pH sensitivity. They incorporated this lipid into the above mentioned MEND siRNA delivery system, and found that the YSK05–MEND combination had a better ability for endosomal escape than other MEND-containing conventional cationic lipids.^[69,70] Some other new cationic lipids have also been reported for improving siRNA delivery, such as N',N'-dioctadecyl-N-4, 8-diaza-10-aminodecanoylglycine amide^[71] and 1,2-dilinoleyloxy-3-dimethylaminopropane, which will be the subject of later discussion.^[23]

Active Targeting Delivery. Incorporating an active target motif, such as transferrin, RGD, EGF and various cell-penetration peptides, to the cationic lipid-based siRNA delivery system could significantly improve the intracellular uptake and endosomal release of siRNA. Daka et al.^[72] and Ogris et al.^[73] have reviewed this aspect extensively.

Example of an Advanced Cationic Lipid-based siRNA Delivery System: Stable Nucleic Acid Lipid Particles & Their Application

Among the cationic lipid-based siRNA vehicles, several advanced delivery systems have gained worldwide attention owing to their high efficiency in systemic siRNA delivery methods, such as LPD, AtuPLEX, stable nucleic acid lipid particle (SNALP) systems, LPD and AtuPLEX, and have been well reviewed previously.^[74,75] Here, we take SNALP as an example to highlight its approach in overcoming barriers of siRNA systemic delivery.

SNALP & its Application. SNALP is constructed by three basic lipids: an ionizable cationic lipid (1,2-dilinoleyloxy-3-dimethylaminopropane), a neutral helper lipid, including Chol and fusogenic lipids, and a PEG lipid (Table 2). In SNALPs, the backbone-modified siRNA is encapsulated within a closed shell of a cationic-zwitterionic lipid bilayer, furnished with an outer PEG shield (Figure 2A). The lipid bilayer contains a mixture of cationic and fusogenic lipids to enable cellular uptake and further endosomal escape owing to electrostatic interactions between the negatively charge cell membrane and cationic lipids. The coated PEG provides a neutral, hydrophilic exterior to shield the cationic bilayer, protect the nucleic acid core against degradation by nucleases, sterically stabilize the particles against disassembly in collagen networks and prevent nonspecific binding to cells, thus making NPs 'cohesive' and 'stealthy' to prevent siRNA degradation and rapid clearance in the bloodstream.

The SNALPs are approximately 70–150 nm in diameter (Figure 2B), and their major accumulation (±standard deviation) was found in mouse liver (28 ± 1.7% of injected dose administered) and spleen (8.2 ± 2.8% of injected dose administratered).^[23] Their biodistribution is related to the property of NPs, such as size, lipid and PEGylation features. In general, the PEGylated NPs are readily navigated to the mononuclear phagocyte systems, such as the liver and spleen. The therapeutic efficacy of SNALP–siRNA against HBV was validated in a HBV replication mouse model by Morrissey et al..^[23] They found that SNALP delivery prolonged the circulation time of siRNA and achieved efficient inhibition of serum HBV by daily intravenous injections of 3 mg/kg/day of siRNA.^[23] Zimmermann et al. further demonstrated the efficient systemic delivery of SNALP–siRNA in nonrodent species. They administered intravenous injections of SNALP–siRNA against the ApoB gene at single doses of 1 or 2.5 mg/kg on cynomolgus monkeys and achieved potent gene knockdown of ApoB mRNA (>90%). Significant downregulation of ApoB protein, serum Chol and low-density lipoprotein (LDL) levels was also observed as early as 24 h after treatment and lasted for 11 days with a 2.5-mg/kg siRNA dose.^[76]

More recently, Semple et al. found a high-performance lipid, 2,2-dilinoleyl-4-dimethylaminoethyl-(1,3)-dioxolane by screening various ionizable cationic lipids, and demonstrated the excellent activity of the 2,2-dilinoleyl-4-dimethylaminoethyl-(1,3)-dioxolane-based SNALP in effectively silencing the endogenous hepatic gene at doses of siRNA as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates in vivo.^[24] Meanwhile, Tao et al. developed another optimized SNALP system by screening cationic lipids and adjusting PEG lipid density, and achieved potent gene knockdown in the liver (90%) of mice.^[77]

Besides the application of SNALP in liver diseases, Judge et al. demonstrated the successful delivery of siRNA to solid tumors in mice targeting the PLK1 gene, resulting in significant inhibition of subcutaneous Hep3B tumor growth.^[78]

Notably, the SNALP system encapsulated with self-amplifying RNA has been reported as a new vaccine platform by Geall et al., which substantially increases immunogenicity by eliciting broad, potent and protective immune responses.^[79] Altogether, it is not surprising that the SNALP system has entered or completed multiple clinical trials for systemic siRNA delivery, such as VEGF and KSP in liver cancer, ApoB in liver disease, TTR in transthyretin amyloidosis, PLK1 in cancer and Elola in Ebola virus disease.^[75]

The impact of physiological constrains, such as tumor vasculature, on the efficiency of siRNA delivery has also been investigated. Li et al. found that SNALP predominantly delivered siRNA to areas adjacent to functional tumor blood vessels by analyzing the spatial distribution of localized target knockdown within tumor sections relative to tumor hypoxia.^[80] This phenomenon suggests that it is probably not easy for the SNALP–siRNA complex to cross the ECM with a size of 70–150 nm; therefore, most of them are trapped in the areas adjacent to blood vessels.^[80] To improve the transport of SNALP across the ECM barrier, Rudorf et al. developed a small SNALP, called a 'mono-NALPs', which was self-assembled by solvent exchange from solution containing siRNA mixed with the four lipid components DOTAP, DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-2000. The mono-NALP has a similar core–shell structure as SNALP, but is only 30 nm in diameter, which should facilitate its transport in vivo across the ECM to achieve deeper tissue penetration.^[81]

Recently, the mechanism of cellular uptake, intracellular transport and the endosomal escape of siRNA by SNALP delivery had been investigated. It has been shown that SNALPs entered cells by both constitutive and inducible pathways in a cell type-specific manner using CME and macropinocytosis (Figure 2C).^[82] However, the escape of siRNAs from the endosome into the cytosol occurred at low efficiency (<5%), suggesting further optimization of the SNALP formulation is required to improve the endosomal release of siRNA (Figure 2D).

Nanomedicine. 2014;9(1):105-120. © 2014  Future Medicine Ltd.