Stable RNA nanoparticles as potential new generation drugs for cancer therapy☆
Graphical abstract
Introduction
Nanotechnology refers to the creation and application of materials using either a top-down approach or bottom-up assembly at the nanometer scale. In nature, a wide variety of macromolecules that form patterned arrays and highly-ordered structures in nano-scale have inspired several biomimetic strategies. Macromolecules, such as DNA, RNA, and proteins have intrinsically defined features with the potential to serve as versatile building blocks for bottom-up assembly of nano-structures and nano-devices.
More and more evidence has revealed that a substantial part of ~ 98.5% of the human genome, so‐called “junk” DNA [1], codes for noncoding RNAs. These noncoding RNAs play major roles in gene expression [2], [3], [4], gene regulation [5], [6], cellular catalytic reaction [7], and so on [8]. The malfunction of some noncoding RNAs will end up as abnormal cellular activity closely related to cancers, for example, microRNAs (miRNAs) have been shown to function as oncogenes or tumor suppressors [9], [10], [11], [12], [13]; and snoRNAs (SNORD33, SNORD66, and SNORD76) were identified as biomarkers for non-small cell lung cancer [14]. Many other diseases, such as dilated cardiomyopathy and heart failure [15], were found to be related to RNA functionality. This has led to treatment strategies that use RNA as therapeutic targets [16], [17]. In other aspects, the discoveries of small interfering RNAs (siRNAs) [18], [19], ribozymes [20], [21], riboswitches [22], [23], and miRNAs [24], [25] have induced a heightened interest in using RNAs as therapeutics for disease treatment.
Natural RNA possesses versatile sequences, secondary structures, and tertiary/quaternary interactions [26], [27], [28]. Several assembly mechanisms of naturally occurring RNA complexes have been applied to construct RNA nanoparticles with defined structure and stoichiometry via intra- and/or inter-molecular interactions. Through this innovative approach based on RNA nanotechnology [29], [30], varieties of therapeutic RNA nanoparticles harboring multiple therapeutic modules, such as siRNA, aptamer, or miRNA, have been constructed. Each incorporated siRNA, aptamer, miRNA, or other functionalities within the nanoparticle fold into its respective, authentic structure and retain its independent function for specific cell binding, cell entry, gene silencing, catalytic function, in both in vitro and animal trials [31], [32], [33]. Following the two milestones of chemical and protein drugs, respectively, in medical history, we speculate that the third milestone in drug development will be RNA drugs or drugs that target RNA, thus, RNA nanoparticles have the potential to be a new generation of drugs. This review will discuss the application of the achievements in modern RNA nanotechnology for cancer therapy, especially focusing on well-constructed pRNA-based RNA nano-delivery systems.
Section snippets
Definition of RNA nanotechnology
RNA nanotechnology is a unique field that studies the design, fabrication, and application of RNA nanoparticles with architectures primarily made up of RNA via bottom-up self-assembly [29], [30], [34], [35] (Fig. 1). This concept contrasts with other widely studied drug delivery nano-systems that conjugate functional RNA modules to polymers, lipids, dendrimers, gold, or other nanomaterial-based particles.
Proof-of-concept of RNA nanotechnology in 1998
Compared to classical RNA studies, RNA nanotechnology is a relatively new field [36], [37], [38], [39], [40], [41]. The first evidence showing that RNA nanoparticles can be constructed by bottom-up self-assembly using reengineered RNA molecules was reported in 1998 [36] (Fig. 2A). The study, led by Peixuan Guo, demonstrated that dimeric, trimeric, and hexameric RNA nanoparticles can be constructed via bottom-up assembly using re-engineered RNA fragments derived from a viral RNA (pRNA) that
Overcome the first barricade: chemical instability of RNAs
Natural RNA is extremely sensitive to RNase degradation and is especially unstable in blood plasma. Over the last few years, rapid progress has been made to improve the stability of RNA for in vivo application by chemical modification of RNA. These include modifications on the bases (e.g. 5-BrU and 5-IU)[78], modifications of the phosphate linkage (e.g. phosphothioate and boranophosphate)[79], alteration of the ribose 2′ hydroxyl group (e.g., 2′-F, 2′-OMe, or 2′-NH2) [53], [80], [81], [82],
Overcome the second barricade: thermodynamic instability of RNA via self-assembly without covalent linkage
The thermodynamic stability of RNA nanoparticles with regard to the use of RNA nanoparticles as therapeutics is of paramount importance. Systemic injection of several micro-liters of RNA solution into the body will result in a hundred-thousand fold dilution. Dissociation of assembled RNA nanoparticles at extremely low concentrations is a serious concern. Crosslinking agents, such as psoralen [91] and transition metal compounds [92] can promote the formation of stable RNA complexes. In a recent
Combating the third barricade: low yield and high production costs
The most challenging aspect of applying RNA nanoparticles for clinical applications is the yield and cost of RNA production. RNA strands can be synthesized both chemically and enzymatically. Commercial RNA chemical synthesis can only offer from up to 40 nucleotide (nt) (conservative) to up to 80 nt (with low yield and high cost). Several methods have been explored for synthesizing longer RNA strands. We found that up to 117 nt of phi29 pRNA can be synthesized using bipartite approach, and this
Advantages of using RNA nanoparticles for pharmaceutical applications
RNA nanotechnology recently has received more and more attention from scientists around the world due to its high potential regarding therapeutics, especially after overcoming aforementioned several major hurdles in the field. While the use of RNA for therapeutics is still in its infancy, it is already clear that RNA nanotechnology provides several advantages: 1) The nano-scale size and branched, ratchet shape of RNA nanoparticles facilitates passive targeting through tumors and narrow cavities
Comparing RNA nanoparticles with other nano-delivery systems
Several nano-delivery systems of different materials and physiochemical properties for the treatment of cancer and viral infections have been pursued (Fig. 3). They include lipid-based nanoparticles [105], polymer-based nanoparticles [106], viral nanoparticles [107], [108], inorganic nanoparticles [109], [110], and DNA nanoparticles [111], [112].
RNA modules applied for cancer therapy
Conventional cancer therapy, including chemotherapy and radiotherapy, cannot distinguish malignant from non-cancerous organs and tissues. Severe side effects and toxicity have occurred in patients during the treatment regime. As such, specific target delivery is highly desired for advanced cancer therapy in order to achieve higher treatment efficacy but lower toxicity than that for conventional therapy. The RNA nanoparticle is one of the candidates for targeting delivery of therapeutics to
Methods for chemical modification/conjugation of RNA nanoparticles
RNA oligonucleotides can be chemically conjugated with many biological or chemical molecules [31], [32]. A well-labeled construct consists of three components: a small molecule moiety, a spacer, and a reactive group (Fig. 4A). The small molecule moiety includes a reporter molecule (fluorophore), any kind of bioactive drug, or ligand. The spacer separates the small molecule moiety from the oligonucleotide and can be used to change the hydrophobicity or hydrophilicity of the molecule and alter
Methods and current achievements for constructing RNA nanoparticles
The general principles of constructing RNA nanoparticles follow three steps: RNA building block extraction, rational computational design/modeling, and RNA nanoparticle fabrication. There are a lot of naturally occurring or artificial RNA motifs that can serve as the building blocks for the design and construction of a variety of RNA nanoparticles.
Challenges and perspectives
The utilization of RNA nanotechnology for medical and nanotechnological applications requires addressing the following issues.
The first challenge is the understanding of correct global folding of RNA constructs to ensure the function of the resulting RNA nanoparticles. RNA folding and structural computation is essential for the examination of new structural designs [61], [66], [67], [68], [69], [197]. Several online resources have been developed, including Mfold [179], RNA designer [198], Sfold
Conclusion
Natural or chemical synthetic RNA molecules can fold into pre-defined structures that can spontaneously assemble into nanoparticles with multiple functionalities. The field of RNA nanotechnology is still emerging, but will play an increasingly important role in medicine, biotechnology, synthetic biology, and nanotechnology. RNA nanoparticles are promising as a new generation of drug for cancer therapy.
Acknowledgments
The authors would like to thank Jennifer Rogers and Jeannie Haak for editing and formatting this review. The laboratory research was supported by NIH grants U01 CA151648, R01 EB003730 to PG, who is a co-founder of Kylin Therapeutics, Inc., and Biomotor and Nucleic Acids Nanotech Development, Ltd. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. Funding to Peixuan Guo's Endowed Chair in Nanobiotechnology is through the William
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This review is part of the Advanced Drug Delivery Reviews theme issue on “Cancer Nanotechnology”.