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Small interfering RNA (siRNA) is an effective method for regulating the expression of proteins, even “undruggable” ones that are nearly impossible to target through traditional small molecule therapeutics. Delivery to the cell and then to the cytosol is the primary requirement for realization of therapeutic potential of siRNA.
We summarize recent advances in the design of inorganic nanoparticle with surface functionality and physicochemical properties engineered for siRNA delivery. Specifically, we discuss the main approaches developed so far to load siRNA into/onto NPs, and NP surface chemistry engineered for enhanced intracellular siRNA delivery, endosomal escape, and targeted delivery of siRNA to disease cells and tissues.
Several challenges remain in developing inorganic NPs for efficient and effective siRNA delivery. Getting the material to the chosen site is important, however the greatest hurdle may well be delivery into the cytosol, either through efficient endosomal escape or by direct cytosolic siRNA delivery. Effective delivery at the organismic and cellular level coupled with biocompatible vehicles with low immunogenic response will facilitate the clinical translation of RNAi for the treatment of genetic diseases.
RNA interference (RNAi) is a specific endogenous gene silencing pathway in which small RNA mediate the degradation of targeted mRNA (Figure 1) [1–3]. RNAi has been extensively utilized in the past decade [3,4] as a powerful tool for selectively controlling protein expression, even proteins that are normally considered to be nearly impossible to target using traditional small molecule therapeutics . Previous research has successfully utilized RNAi as a tool for examining downstream effects from the knockdown of particular genes [ 6 – 8 ]. Therapeutic delivery of siRNA has also been shown to be effective in treating diseases like cancer [9, 10]. However, there are many hurdles to overcome before realizing the therapeutic potential of RNAi. In particular, siRNA is an unstable negatively charged biomacromolecule and cannot effectively cross cell membranes, thus requiring a delivery vehicle [4, 11, 12]. Traditionally, siRNA was introduced into cells via physical methods such as microinjection and electroporation [13 ]. However, these methods are not suitable for most in vivo uses. Virus-based delivery strategies are effective for delivery of siRNA [14, 15], however they can induce insertional mutagenesis and have undesired immune response . In recent years as RNAi research has shifted from fundamental studies towards applications for disease treatment, new methods of introducing siRNA are required.
A large number of non-viral vehicles have been developed for siRNA delivery, including cationic polymers , liposomes , virus-like particles , cell penetrating peptides , and inorganic nanoparticles [ 21 ]. Despite numerous efforts, very few efficient and effective approaches for siRNA delivery have emerged. The challenge is multifaceted: first the vehicle must effectively cross the cell membrane while avoiding or escaping endosomal entrapment [22, 23]. The vector requires stability outside the cell, in particular from degradation by nucleases , but then effectively release the payload inside the cell.
Inorganic nanoparticles provide useful platforms for the engineering of RNA-based delivery vehicles for in vitro and in vivo applications [12, 23, 24–29]. Inorganic NPs have high surface area to volume ratio, allowing for efficient loading of siRNA via direct conjugation or non-covalent encapsulation. A key advantage of these NPs is that their surface chemistry is easily modified, providing a means of overcoming the challenges of in vitro and in vivo siRNA delivery . In addition, the unique physical and optical properties of inorganic NPs can be used to track siRNA delivery to cells or tissues [30, 31]. There have been a wide range of inorganic NPs used for delivery (Table 1), and their advantages and disadvantages compared to organic NPs-based siRNA delivery system was summarized in Table 2 [6, 9, 20, 21, 24]; this review will focus on recent advances that we feel provide useful leads for therapeutic development.
AuNPs have physical and chemical properties that make them perhaps the most studied inorganic NPs for siRNA delivery [27, 29, 32]. First, the core of AuNP is essentially inert, non-toxic, and biocompatible, thus enabling a stable and safe siRNA delivery platform. Second, the well-developed surface chemistry of AuNPs allows the conjugation of siRNA via either covalent or non-covalent chemistry. Finally, the ability to “tune” AuNP properties through surface modification provides useful properties for controlling NP delivery and release.
A common approach for siRNA delivery involves the covalent conjugation of siRNA with various cell-penetrating or targeting ligands, such as lipids , peptides , and small molecule receptors . These conjugates have shown effectiveness for siRNA delivery, with GalNAc-conjugated siRNA exhibiting promising results in silencing targeted gene in clinical trials . However, these siRNA conjugates usually lack serum stability and have a short circulation time which prohibits a long term gene silencing effect. NP-based siRNA delivery approaches have shown superior stability against enzymatic degradation, and additionally prevent the premature release of siRNA into the physiological environment that can occur using other siRNA conjugates.
The most direct method to use AuNPs for siRNA delivery is to conjugate siRNA onto AuNP surface via thiol-gold covalent chemistry. Nagasaki et al. first used siRNA–AuNP conjugates for siRNA delivery in 2006 . They modified 15 nm AuNPs with thiol–PEG5000–PAMA7500 polymer, followed by self-assembling thiolated siRNA. The resulting AuNP/siRNA conjugates led to a 65% gene silence efficiency with human hepatoma HuH-7 cells. Mirkin et al. has extended this concept to create spherical nucleic acid (SNA) nanoparticles (Figure 2a). SNAs are remarkably stable in physiological environments and resist nuclease degradation. They first conjugated13 nm AuNPs with thiol-siRNA and oligoethylglycol thiol . The use of a shorter PEG higher than 99% . In subsequent studies, SNAs against Bcl2Like12 (Bcl2L12) oncogene were effective in knocking down endogenous Bcl2L12 mRNA and protein levels of glioma cells [ 36 ]. The systemic administration of SNAs reduced Bcl2L12 expression in intracerebral glioblastoma multiforme, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice. More recently, the same group demonstrated that SNAs dispersed in Aquaphor can penetrate the epidermal barrier of both intact mouse and human skin, enter keratinocytes, and efficiently silence target genes resulting in enhanced rates of wound healing .
Covalent siRNA/AuNP conjugates can be further coated with cationic polymer or cell penetrating peptides to enhance siRNA delivery efficiency. For example, Reich et al. first conjugated thiolate siRNA onto a gold nanoshell surface, followed by coating the NP surface with a streptavidin layer used to attach cell penetrating peptides through biotin-streptavidin ligation . Upon irradiation with biocompatible near infrared (NIR) light, the siRNA was released from the gold surface and escaped from the endosome. The siRNA construct was efficiently internalized by a broad range of human embryonic stem cells (hESC), knocking down of Oct4 induced the differentiation of hESC to all three germ layers. Anderson et al. conjugated thiol-siRNA onto amine-coated AuNP using a dual-functional crosslinker, succinimidyl 3-(2-Pyridyldithio)Propionate (SPDP) [ 39 ]. The siRNA-AuNP conjugates were post-coated with biodegradable polymer, poly (β-amino ester), to improve siRNA gene silencing efficiency. The treatment of luciferase-expressing HeLa cells with 90 nM siRNA silenced gene expression in an efficiency higher than 90% in serum-containing medium.
The ease of functionalization of AuNPs makes them excellent platforms for targeted delivery vehicles. For example, the simultaneous conjugation of thiol-siRNA and Arg-Gly-Asp (RGD) onto AuNP efficiently delivered siRNA to the tumor site in a lung cancer orthotopic murine model . Knocking down c-myc oncogene using this targeting siRNA/AuNP conjugate resulted in tumor growth suppression and prolonged survival of lung tumor bearing mice.
The simplicity and versatility of the AuNP scaffold allows the creation of complex machinery. Kim et al. reported an i-motif-driven gold nanoparticle-based nanomachine for programmed siRNA delivery . They immobilized a pH-responsive nucleic acid consisted of three functional segments, i.e., an i-motif DNA, which has an acid-responsive switchable secondary structure, an overhanging linker DNA, and a therapeutic siRNA, on the nanomechine. After entering cells, the acidic endosome environment triggers the formation of an interstrand tetraplex of i-motif to induce cluster formation of AuNP, which facilitates endosomal escape and releases siRNA into cytosol. The delivery of siRNA against polo-like kinase 1 (PLK1) significantly reduced the viability of NIH3T3 cells, suggesting the therapeutic potential of this strategy.
Non-covalent delivery strategies provide a versatile modular strategy for the delivery of free siRNA. The negative charge of the siRNA strand facilitates assembly with positively charged NPs. For example, Rotello et al. have reported dendronized AuNPs for siRNA delivery (Figure 2b) [ 42 ]. AuNPs (2 nm core) were functionalized with dendronized ligands based on biodegradable glutamic cores with cationic triethylenetetramine (TETA) termini. The second-generation of dendronized AuNPs (G2-AuNPs) encapsulated siRNA with the highest efficiency. G2-AuNP delivered β-galactosidase targeting siRNA (β-gal–siRNA) into SVR-bag4 cells, with knockdown efficiency up to 50%, as measured by enzyme activity assay.
Cationic polymers, such as PEI and polylysine (PLL), have a long history as gene delivery vehicles. The direct functionalization of AuNP with these polymers enables encapsulation of siRNA and delivery. For example, Park et al. reported the controlled synthesis of PEI-coated AuNP using catechol-conjugated PEI for siRNA delivery . Also, PEI has been used as both reductant and stabilizer to synthesize AuNPs which have been used to silence GFP and oncogenic PLK-1 gene via siRNA delivery for cancer therapy .
Layer-by-layer (LBL) coating of a gold core with cationic polymers and siRNA is another method for creating polymer-coated AuNPs for siRNA delivery. Negatively charged AuNPs were complexed with bPEI (25 kDa) and then sequentially deposited siRNA and bPEI to form AuNP/bPEI/siRNA/bPEI complexes, which showed an increased hydrodynamic diameter of 22 ~ 25 nm. On average, around 780 siRNA molecules were complexed with each AuNP, which was a significant enhancement compared to covalent siRNA-AuNP conjugates. The gene silencing efficiency of AuNP/bPEI/siRNA/bPEI complexes was verified by delivering siRNA against EGFP to CHO-K1 cells . Hahn reported LBL assembled cysteamine (CM) modified AuNP (AuCM)/siRNA/PEI/hyaluronic acid (HA) for intracellular delivery of siRNA . The introduction of HA into the NPs enabled the targeted delivery of siRNA to B16F1 cells which has HA receptors. The authors demonstrated the capability of (AuCM)/siRNA/PEI/HA for in vivo siRNA delivery by knocking down apolipoprotein B expression in mouse liver.
The use of biodegradeable biopolymers provides a means of reducing the toxicity of the carrier. For example, Tung et al. incorporated protease-degradable poly-lysine (PLL) and siRNA on the surface of AuNPs, and reported siRNA/PLL multilayer-coated AuNPs for siRNA delivery . The PLL multilayer can be degraded by lysosomal cathepsin B enzyme to release siRNA gradually, inducing a prolonged gene-silencing effect. They later demonstrated that AuNPs with two layers of siRNA and three layers of PLL provided 20 days of > 60% luciferase knockdown in a MDA-MB-231-Luc xenograft .
More recently, “charge-reversal” polyelectrolytes have been applied to siRNA delivery. The “charge-reversal” polymer change net charge, from positive at neutral pH to negative under acidic environment efficiently controls siRNA release inside cells. For example, Liang et al. have used pH-responsive cis-aconitic anhydride-functionalized poly(allylamine) (PAH-Cit), and developed a “charge-reversal” LBL AuNP/siRNA delivery method (Figure 2c) . AuNPs were initially modified with negatively charged 11-mercaptoundecanoic acid (MUA), upon which bPEI was deposited forming a cationic AuNP-MUA/bPEI core for LBL assembly. PAH-Cit and bPEI were then sequentially deposited onto the AuNP-MUA/bPEI core to encapsulate siRNA. The assembled PEI/PAHCit/PEI/MUA-AuNP/siRNA shows improved endosome escape efficiency and a higher gene knockdown efficiency than commercial lipid reagents and bPEI. Alternatively, charge-reversal chitosan (CS) can assemble similar LBL AuNP to deliver siRNA that silences the multidrug resist gene MDR1 , showing a potential pathway to overcome the drug resistance problem of traditional chemotherapy.
A key barrier to efficient siRNA delivery and effective RNAi is the delivery of siRNA cargo to the cytosol, where siRNA is loaded into the RISC to degrade mRNA. Nanomaterials usually enter cells via an endocytic pathway, resulting in endosome entrapment. It has been reported that even with relatively efficient vectors only a small fraction (1–2%) of internalized siRNA can escape from the endocytic pathway . Designing nanocarriers that facilitate endosome escape of siRNA, or directly deliver siRNA into cytosol is therefore highly desired for effective RNAi [23, 52].
One commonly used method to facilitate endosomal escape of siRNA is to design highly positively charged polyamine carriers, which enhance endosome escape capability through the “proton sponge effect” . These polyamine carriers, such as PEI and its derivatives, sequester protons when they enter the acidic lysosome compartment of cells due to the large buffering capacity of secondary or tertiary amines presented on the carriers. The continuous protonation of polyamine carriers subsequently pump chloride ions into the endosome, causing a water influx that leads to endosomal swelling and lysis, releasing siRNA into the cytoplasm [22, 23]. However, a recent study revealed that the widely used PEI played a limited role on enhancing endosome escape even under strong acidic lysosomal environment . Introducing membrane-disruptive or fusogenic peptides such as GALA and KALA [54, 55], onto AuNP surface improves the endosomal escape of siRNA to certain extent, with improved gene knockdown efficiency. Alternatively, hollow AuNPs that can be heated with NIR light irradiation have facilitated escape of siRNA from the endosome. For example, Li et al. demonstrated that conjugates of siRNA targeting NF-KB p65 and hollow AuNP allowed for controllable cytoplasmic siRNA delivery upon NIR light irradiation .
A different strategy is to avoid endosomal uptake altogether. Rotello et al. recently developed a platform that provides direct cytosolic delivery of siRNA using nanoparticle-stabilized nanocapsules (NPSC) (Figure 3) . In this approach, siRNA is complexed with cationic arginine-functionalized gold nanoparticles via electrostatic interactions, with the resulting ensemble self-assembling onto the surface of fatty acid nanodroplets to form a NPSC/siRNA nanocomplex. The complex rapidly delivers siRNA into cytosol in a cholesterol-dependent manner via membrane fusion. Live cell images and video show fluorescently-labeled siRNA was evenly distributed within the whole cell, with no punctate fluorescence indicative of endosomal uptake. This method delivered siRNA and silenced destabilized green fluorescent protein (deGFP) of HEK293 cells up to 90%. Moreover, the delivery of siRNA targeting PLK1 efficiently silenced oncogenic PLK1 expression in cancer cells showing the potential of this system for cancer therapy.
Magnetic nanoparticles (MNPs) provide useful properties for siRNA delivery. First, external magnetic fields can be used to manipulate the MNPs for spatially controlled siRNA delivery. Second, MNPs can be exploited with magnetic resonance imaging (MRI) to track siRNA delivery. Both of these properties were employed by Cheon et al., who generated multifunctional MNPs (MnMEIO) by conjugating siRNA onto a MNPs surface. They first coated MNPs with serum albumin (BSA), followed by conjugation of fluorescent and thiolated siRNA using SPDP as a crosslinker (Figure 4a) . In addition, an RGD peptide was introduced to target αvβ3 integrin overexpressing metastatic tumor cells. The targeted delivery of siRNA into αvβ3 integrin overexpressed MDA-MB-435 cells was verified by T2*-weighted MR imaging. MnMEIO/siGFP/RGD nanoparticle efficiently knocked down GFP expression in MDA-MB-435 cells, while no GFP knockdown was observed for αvβ3 integrin negative A549 cells under the same conditions. Similarly, Medarova et al. designed “all-in-one” MNPs for siRNA delivery and tumor imaging . NIR fluorophore Cy5.5 and a peptide targeting tumor-specific antigen uMUC-1, were simultaneously conjugated to MNPs, enabling the visualization of siRNA delivery to tumor sites using MRI and NIR optical imaging.
Similar to AuNPs, surface modification of MNPs using cationic polymer and lipids allows electrostatic encapsulation of siRNA. Chen et al. coated magnetic iron oxide nanoclusters with cationic alkylated PEI, and used these nanoparticles for siRNA delivery in vitro and in vivo using an fLuc-4T1 xenograft model to demonstrate luciferase knockdown [ 60]. Magnetic nanocrystal clusters (PMNCs) crosslinked with PEI provide an alternative magnetic platform for siRNA delivery in vitro [ 61 ]. Bhatia et al. developed dendrimer-conjugated magnetofluoresent nanoworms, called “dendriworms” for siRNA delivery in vivo [ 62 ]. These siRNA-carrying dendriworms were internalized by cells with high endosomal escape and EGFR knockdown of 70–80%. Anderson et al. reported a simple method to load and deliver siRNA by coating iron oxide nanoparticles with lipid-like molecules, called lipidoid . The combinatorial synthesis of lipidoids allows the generation of a library of lipid-coated MNPs and for screening of the most effective siRNA delivery vehicles. One of the top lipid candidates for siRNA delivery, C14–200 coated MNPs, delivered siRNA and silenced luciferase expression of modified HeLa cells up to 80% at a siRNA concentration as low as 1.5 nM.
Magnetically-guided delivery provides a tool for spatial control of therapeutics. Namiki and co-workers assembled oleic acid-coated magnetic nanocrystals with cationic lipid formulations to generate LipoMag particles for siRNA delivery . Intravenous injection of LipoMag, combined with the application of an external magnetic field, resulted in 50% reduction of tumor growth in gastric tumor bearing mice.
Mesoporous silica nanoparticles (MSNs) have been used to deliver a variety of drugs, including small molecules, genes, and proteins . The large surface area of MSNs provides space and binding sites to accommodate siRNA via non-covalent encapsulation or direct covalent conjugation. For example thiolated siRNA was efficiently conjugated to MSN surfaces for delivery. Also, coating MSNs with cationic polymers (e.g., polyethyleneimine, polyamidoamine, and polylysine) has been demonstrated to be efficient in encapsulating siRNA via electrostatic interactions [66, 67, 68]. Nel et al. coated MSNs with PEI and studied the effect of polymer size on cellular uptake of MSNs. They found that 10 KD PEI-coated MSNPs were capable of delivering siRNA and knocking down GFP expression of modified HEPA-1 liver cancer cells without cytotoxicity. . In another approach, Gu et al. encapsulated siRNA into the mesoporous pores of MSNs, followed by surface coating with PEI and a fusogenic peptide (KALA) to enhance the endosomal escape of MSNs . The resulting MSN/siRNA complex protected siRNA from degradation, had low toxicity, and was efficiently internalized by cells. The efficacy of this system was demonstrated by delivery of siRNA targeting vascular endothelial growth factor (VEGF) into tumor-bearing mice, effectively suppressing tumor growth. To reduce the cytotoxicity associated with the use of direct b-PEI-coated MSNs for delivery, Lellouche et al. used cerium (III) cations (Ce3+) to crosslink bPEI to poly-amine modified MSNs. It was concluded that Ce3+ -nitrogen (from the amines of MSNs and PEI) play critical roles in attaching the bPEI shell onto MSNs surfaces for siRNA delivery .
The coating of PEI onto MSNs surface provides additional primary amine groups to conjugate targeting ligands for cancer cell targeted siRNA delivery. For example, anti-HER2 monoclonal antibody (trastuzumab)-conjugated PEI/MSNs selectively deliver siRNA into HER2-positive cells . The delivery of siRNA selectively induced apoptotic death of HER2-positive BT474 cells, but not HER2-negative MCF-7 breast cancer cells. The administration of targeted PEI/MSNs/siRNA (against HER2 oncogene) into orthotopic HCC1954 tumor bearing mice effectively suppressed tumor growth.
The pores of MSNs allow the accommodation of small molecules, while the MSNs surface can be engineered with cationic polymers to encapsulate siRNA via electrostatic binding, providing a vehicle for co-delivery of a drug and siRNA for synergistic therapy. Chen et al. designed a MSN-based co-delivery method by loading doxorubicin into large capacity MSNs . The MSN exterior was then modified with G2 amine-terminated polyamidoamine (PAMAM) dendrimers for siRNA encapsulation. The co-delivery of doxorubicin and siRNA targeting Bcl-2 gene to MDR cancer cells significantly restored the sensitivity of the cells toward doxorubicin treatment, compared to that without siRNA delivery. Coating of MSNs with folic acid (FA) enabled the co-delivery of doxorubicin and Bcl-2 siRNA into FA-positive HeLa cells with enhanced therapeutic efficiency . Finally, Zink et al. demonstrated that the co-delivery of doxorubicin and siRNA against P-glycoprotein (Pgp) drug exporter silenced P-gp expression in a MCF-7/MDR xenograft model in nude mice, resulting in a synergistic inhibition of tumor growth in vivo (Figure 4b) .
Calcium phosphate (CaP) is a natural inorganic material found in human bone and teeth with promise for drug delivery due to its biocompatibility and biodegradation ability. CaP can encapsulate negatively charged gene cargos through the chelation of calcium ions and the phosphate of genes, forming CaP nanoparticles that are internalized by cells . However, CaP-based siRNA delivery currently has challenges arising from the low endosome escape efficiency of CaP and uncontrollable growth of CaP/siRNA NPs under physiological environment.
Kataoka et al. self-assembled a pH-responsive block polymer, poly(ethylene glycol)-block-poly(methacrylic acid) (PMA), into CaP/siRNA NPs to enhance endosomal escape of CaP vehicles. PMA becomes more hydrophobic under an acidic environment (pH = 4–6), disrupting the endosome membrane and releasing siRNA . The authors report a high gene knockdown efficiency using PMA/CaP/siRNA NPs to silence luciferase in modified human embryonic kidney 293 cells. Similarly, Jeong et al. the natural anionic polymer hyaluronic acid (HA), conjugated with dioleoylphosphatydic acid (DOPA) to control the size and stability of CaP/siRNA NPs (Figure 5a) . The CaP/siRNA/DOPA-HA NPs maintain the integrity and stability of encapsulated siRNA, and showed improved intratumoral accumulation of siRNA and a high level of target gene silencing in solid tumors after systemic administration. Huang et al. used an alternate strategy for stabilizing CaP nanoparticle, using an asymmetric lipid bilayer for tumor targeted siRNA delivery . PEGlyation and functionalization of the above CaP NPs with targeting anisamide ligands enabled the delivery of siRNA to human H460 lung cancer xenografts with improved in vivo gene silencing efficiency.
Inorganic nanoparticles provide a diverse set of platforms for siRNA delivery. The broad range of chemical approaches for engineering NP surfaces provides the ability to further optimize siRNA delivery in vitro and in vivo. In addition, the unique physical and optical properties of inorganic NPs have been used to track siRNA delivery and bio-distribution, and for the development of theranostic agents. The therapeutic potential of inorganic NPs for siRNA delivery has been demonstrated in a broad range of disease models, providing promise for future therapeutic applications. However, there are remaining hurdles that are preventing the progression of this delivery mechanism to clinical trials, evidenced by the fact that there are no currently active clinical trials using inorganic nanoparticles for RNAi therapeutics. Engineering and manufacturing siRNA-loaded inorganic nanoparticles with homogenous size, composition, and surface charge in a simple, fast, and inexpensive manner require concerted effort. Likewise, critical information on the safety of siRNA-loaded nanoparticles in regards to injection/administration, clearance route, and potential immune response in long-lasting and sustained release platforms are still unclear. Strategies for diminishing the risks of nanotoxicity, and the methodology for evaluating these strategies need to be carefully designed. Most importantly, the lack of fast and inexpensive screening tools for intermediate and final adducts of functional NP-RNA complexes using pre-clinical animal models has greatly hampered clinical translation. Before these issues can be addressed, however, there is still much to be done in terms of biological barriers at the organismic, tissue, and cellular levels. Overcoming these challenges will require improved understanding of the fundamental aspects of nanomaterial interactions with biological systems, research that is underway world-wide.
RNAi has the potential to revolutionize treatment of a broad range of diseases, particularly “undruggable” targets. We currently have a diverse set of inorganic nanocarriers for siRNA delivery that are effective both in vitro and in vivo. Each of these approaches, however, currently have limitations and challenges that requires further optimization to overcome the barriers present for effective therapeutics.
Toxicology is an important issue, with inorganic NPs generating new and unpredicted behaviors. Most studies of the biocompatibility of inorganic NPs were evaluated using a simple cytotoxicity evaluation; however, detailed toxicological evaluation, such as cell membrane damage, oxidative stress, genotoxicity, are required for predicting long-term effects of NP exposure.
A key challenge for siRNA therapeutics is getting the material efficiently to where it needs to be. This issue is multi-scale, with hurdles at the organismic, tissue, and cellular level. On the organismic level, pharmacokinetics (PK) is an important concern. Effective nanoparticle vectors should minimize the recognition and clearance by the reticuloendothelial system (RES) after systemic administration. Grafting non-interacting functional groups, such as PEG and zwitterionic entities, have proved to be efficient at decreasing NP clearance by RES and increase in vivo circulation. However, these methods also decrease the siRNA delivery efficiency due the PEG shielding of the NPs or by decreasing the intracellular release of siRNA. Clearly, new chemistries for preventing serum protein adsorption onto NPs will improve the pharmacokinetics of NPs, and reduce the NP uptake by RES.
Biodistribution is an issue that will be helped but not solved by non-fouling coatings. The conjugation of targeted ligands to inorganic NPs enhanced the accumulation of siRNA at the disease sites, in particular, tumors. However, the decoration of target ligands on each type of NPs requires careful case-by-case validation for enhanced siRNA delivery. In addition, the conjugation of targeted ligands to NPs may also alter the PK and stability of the vectors, complicating data interpretation of in vivo studies.
Perhaps the greatest hurdle for siRNA therapeutics, however, is avoiding endosomal entrapment and subsequent degradation. This is a crucial issue, as currently delivery to the cytosol is generally remarkably inefficient, with consequent off-target effects arising from the large doses of vector required for knockdown efficacy. Future design of inorganic NPs requires careful consideration and accurate optimization of NP chemical functionalities to improve cytosolic siRNA delivery and avoid off-target effects. For example, installing new functionality onto NPs to modulate siRNA release, using the disease microenvironment or characteristic disease types and stages, could deliver siRNA more efficiently. In addition, integrating optical or other tracking signals to NPs provides new approaches to evaluate the colloidal and surface ligand stability of NPs in vivo, offering feedback for optimizing the design of the nanoparticles to further improve the delivery. Aditionally, chemically modified siRNA has exhibited higher RNA stability and reduced off-target effects compared to traditional unmodified siRNA. As a result NPs that can compensate for the new biophysical structure and biological function of modified siRNA are highly desirable. Finally, to apply these next-generation inorganic NPs in clinical settings, their ‘negative’ effects, such as the nanotoxicity and immunogenicity, should be carefully inspected. A comprehensive study of liver, inflammatory, hematologic, serologic, and kidney toxicity should be determined and correlated to NP structure.
Taken together, the tools that are available for achievements synthesis and surface engineering of inorganic NPs make these systems promising platforms for the very challenging yet rewarding goal of RNAi therapeutics.
Declaration of interest
This research was supported by NIH R01 GM077173, National Distinguished Young Scholars grant (31225009) of Chinese Natural Science Foundation, “Strategic Priority Research Program” of the Chinese Academy of Sciences, as well as t financial support from China Scholarship Council. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed
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