Favorable Biodistribution, Specific Targeting and Conditional Endosomal Escape of RNA Nanoparticles in Cancer Therapy

Abstract

The past decades have witnessed the successful transition of several nanotechnology platforms into the clinical trials. However, specific delivery of therapeutics to tumors is hindered by several barriers including cancer recognition and tissue penetration; particle heterogeneity and aggregation; unfavorable pharmacokinetic profiles such as fast clearance and organ accumulation. With the advent of RNA nanotechnology, a series of RNA nanoparticles have been successfully constructed to overcome many of the aforementioned challenges for in vivo cancer targeting with favorable biodistribution profiles. Compared to other nanodelivery platforms, the physiochemical properties of RNA nanoparticles can be tuned with relative ease for investigating the in vivo behavior of nanoparticles upon systemic injection. The size, shape, and surface chemistry, especially hydrophobic modifications, exert significant impacts on the in vivo fate of RNA nanoparticles. Rationally designed RNA nanoparticles with defined stoichiometry and high homogeneity have been demonstrated to specifically target tumor cells while avoiding accumulation in healthy vital organs after systemic injection. RNA nanoparticles were proven to deliver therapeutics such as siRNA and anti-miRNA to block tumor growth in several animal models. Although the release of anti-miRNA from the RNA nanoparticles has achieved high efficiency of tumor regression in multiple animal models, the efficiency of endosomal escape for siRNA delivery needs further improvement. This review focuses on the advances and perspectives of this promising RNA nanotechnology platform for cancer targeting and therapy.

Introduction

Efficacious responses in patients across a wide range of diseases rely heavily on the bioavailability and delivery of drugs specifically at sites of interest. Cancer represents the best example of a disease where potent yet toxic chemotherapeutics can make the difference between positive outcomes and severe side-effects [1]. Despite significant financial investment, present-day formulations result in significant accumulations in healthy vital organs and target tumors with low specificity. Addressing such issues, nanoparticle-based drug delivery is emerging as a powerful tool to alter the pharmacokinetics and biodistribution of chemotherapeutic and RNAi based drugs administered systemically [2]. By shifting the balance between off- and on-target accumulation, several nanomedicines such as Doxil™ (liposomal doxorubicin) and Abraxane™ (paclitaxel-containing albumin nanoparticles) have gained success in the clinic [3]. Even so, most current drug delivery platforms face limited site-specific bioavailability due to a series of roadblocks associated with formulation challenges (particle heterogeneity, particle aggregation, particle dissociation, high production costs, and unstable thermodynamic and chemical properties); unfavorable biodistribution and pharmacological profiles; and difficulty to overcome biological barriers surrounding tumors [4, 5]. In addition, lack of controlled-release slows down the clinical translation. The complex composition of nanocarriers with diverse functional modules (inorganic/organic nanoscaffolds, RNA/protein antibodies combination, chemical drugs/antibodies complexing) also delays regulatory approval of these nanoparticles [6, 7].

With the advancement of RNA nanotechnology field over the past decade, RNA-based nanoparticles have shown great potential for delivering therapeutics in vivo with favorable pharmacological profiles [8–11]. RNA, as a naturally-occurring polymer, is advantageous as a building block for bottom-up assembly of nanoparticles with defined size, shape, and stoichiometry, composed purely of RNA nucleotides [12]. RNA exhibits both canonical Watson-Crick (A-U, G-C) and noncanonical (such as G-U) base pairing [13] and this property along with base stacking and tertiary interactions enable the RNA to fold into complex structural conformations [28]. Several RNA nanoparticles have been constructed with tunable chemical and thermodynamic properties suitable for in vivo drug delivery [14]. More recently, the three-way junction (3WJ) motif of packaging RNA (pRNA) molecule derived from the phi29 DNA packaging motor have shown great potential in developing RNA nanoparticles for cancer targeting and therapy [9]. The 3WJ nanoparticles can be functionalized with varieties of imaging (fluorophores [15] or fluorogenic aptamers [16, 17]), targeting (RNA aptamers or chemical ligands [18]) and therapeutic (siRNA [19], miRNA, anti-miRNA [18, 20, 21], ribozymes [17] or chemical drugs [22]) modules without affecting the folding of the scaffold or function of the incorporated modules [17]. Upon systemic injection in tumor-bearing mice, the pRNA-3WJ based RNA nanoparticles specifically accumulate at the tumor site. The elastic nature and branched ratchet shape of RNA nanoparticles facilitates tumor penetration and increases EPR (enhanced permeability and retention) effects [23]. This is particularly useful for overcoming mechanical barriers, disorganized vascularization, and highly immunosuppressive tumor microenvironments. Incorporating targeting ligands allows RNA nanoparticles to bind cell surface receptors and enter cells through receptor mediated endocytosis and in the process, deliver therapeutics to the cells. Little or no accumulation of RNA nanoparticles is observed in vital organs. The negative charged backbone of RNA minimizes nonspecific binding to negatively charged cell membrane, thus reducing toxicity and side effects. Moreover, the pRNA-based nanoparticles display favorable pharmacological profiles; are non-toxic; and induce extremely low interferon or cytokine production in mice [19, 24]. These promising results indicate that RNA nanotechnology-based delivery platforms can potentially solve many of the current challenges that other nanoparticles typically face in cancer therapy. Enhanced therapeutic effects of anti-cancer agents are anticipated when using these platforms, and specific delivery to cancer cells will reduce the required dose and associated side effects. The ease of incorporation of RNAi therapeutics will also make the platform ideal to treat cancers by targeting undruggable mutations, tackling drug-resistance, and preventing cancer recurrence. From a translational perspective, RNA nanoparticles are biocompatible, well-characterized, and the production process is relatively easy, scalable, reproducible, and cost effective. Thus, the translation of RNA nanotechnology to a clinical setting is highly likely. Here we provide a brief review focusing on the defining aspects of RNA nanoparticles’ designs and tuning the physiochemical properties of RNA nanoparticles to treat a wide range of cancers using the pRNA-3WJ motif as a shining example.

Construction and characterization of RNA nanoparticles

Methods for the construction of RNA nanoparticles

Over the years, several techniques have been developed for constructing RNA nanoparticles with various shape, sizes, and stoichiometry (reviewed in [8, 10–12, 25]). Briefly, methods include: (1) Loop-loop or hand-in-hand interactions (complementary sequences form interlocking loops) [14, 26, 27]; (2) Foot-to-foot interactions (palindrome sequences mediated self-assembly) [14, 26]; (3) Rational design using RNA motifs, such as kissing loops, dovetails, pseudoknots, kink turns, and multi-way junctions [28–30]; (4) RNA architectonics (rational design of 3D structures from known structures of RNA) [30–35] from databases such as the Nucleic Acid Database (NAD) [36] and RNAjunction database [37]; (5) Computational approaches (computer assisted de novo 3D design) using software programs, such as Nanotiler [38], Assemble2 [39], RNA2D3D [40], INFO-RNA [41], and NUPACK [42]; (6) Co-transcriptional assembly (generate RNA nanoparticles from a DNA template during in vitro transcription) [43–45]; (7) Rolling Circle Transcription (generate concatemeric RNA sequences from a circular DNA template during in vitro transcription) [46, 47]; (8) RNA origami (construction of complex shapes from a single stranded RNA during in vitro transcription) [43].

Synthesis of RNA nanoparticles

RNA nanoparticles typically employ modular design principles, that are composed of multiple strands. Short RNA oligomers (< 80 nucleotides) can be synthesized chemically using an oligo synthesizer based on solid phase phosphoramidite chemistry. Modified nucleotides (such as, 2’-F, 2’-OMe, LNA, etc.) can be incorporated at the desired location in the sequence. Longer RNA strands (> 80 nucleotides) need to be transcribed from a DNA template using T7 RNA polymerase. A limited number of modified nucleotides can be incorporated using a mutant RNA polymerase.

Current chemical synthesis method is only feasible for short RNA strands. Synthesis of RNA with sequence longer than 80 nucleotides will result in low yield. By applying RNA nanotechnology, larger RNA particles can be built via bottom-up assembly using smaller RNA fragments.

Functionalization and labeling of RNA nanoparticles

RNA based ligands, such as, targeting aptamers; therapeutic siRNA, miRNA, anti-miRNA, and ribozyme; and regulatory modules, such as, ribozymes can be rationally incorporated into RNA nanoparticle scaffolds simply by sequence fusion [9, 14, 18, 19, 21, 48–53]. Small molecules, such as, targeting ligands folate and galactose; fluorophores, such as, Alexa647; or chemical drugs can be conjugated to RNA strands using standard chemical reactions (reviewed in [11]). These include: (1) NHS chemistry via coupling of an activated carboxylic group with a primary amine group on an oligo; (2) Bioorthogonal click chemistry between azide group on ligand and alkyne group on an oligo; (3) Thioester linkage using thio-modified oligo and maleimide on ligand; (4) EDC (carbodiimide) crosslinking through amino group and carboxylic acid groups; and, (5) Coupling of a phosphoramidite derivative of the labeling group with the oligo during solid phase synthesis.

Self-assembly of RNA nanoparticles

Typically, the component strands of RNA nanoparticles are mixed in stoichiometric ratio in room temperature or annealed (heated to 95°C and slowly cooled to room temperature). Different from bottom-up assembly, RNA nanoparticles can also be assembled co-transcriptionally [43–45] or through rolling cycle transcription [46, 47].

Purification and characterization of RNA nanoparticles

Individual strands are typically purified by HPLC or denaturing PAGE (polyacrylamide gel electrophoresis). Assembled RNA nanoparticles are generally purified by nondenaturing PAGE, HPLC, or ultracentrifugation [54]. The RNA nanoparticles are then characterized as follows: (1) Assay RNA nanoparticle folding and assembly using native PAGE gels [14]; (2) Assess T_m by qPCR with SYBR Green, temperature gradient gel or UV absorbance [35, 55]; (3) Assess K_d by competition assays using radiolabeled RNA [9] or Surface Plasmon Resonance[30, 56]; (4) Evaluate chemical stability by incubating RNA with RNase or 50% FBS[9, 57]; (5) Examine resistance to Urea by denaturing PAGE [9, 50]; (6) Structural characterization by Atomic Force Microscopy (AFM) imaging [14, 32, 58]; (7) 2D structure prediction by 'mfold' [59].

Novelty of pRNA derived from phi29 DNA packaging motor in the burgeoning field of RNA nanotechnology

The first proof-of-concept demonstrating the feasibility of RNA nanotechnology was published in 1998 in Molecular Cell (featured in Cell) [60]. Dimer, trimer, and hexamer complexes were constructed by bottom-up assembly using reengineered pRNA fragments using hand-in-hand or loop-loop interactions (Fig. 1A). A second approach relied on foot-to-foot interactions to connect two pRNA monomers using palindrome sequence mediated self-assembly [14, 18]. The pRNA monomer contains a 3WJ motif in the central domain (Fig. 1B). More recently, it was shown that the 3WJ motif can be extracted from the core pRNA sequence and used as a scaffold, which represents a third technique for RNA nanoparticle construction [9, 19, 21, 49]. Atomic force microscopy (AFM) characterization of extended 3WJ showed the branched structure, allowing for incorporation of multiple modules (Fig. 1C) [18]. Over the past decade, Guo and co-workers have used these three approaches to design series of RNA nanoparticles with defined size, shape, and stoichiometry.

Figure 1.

Sequence of (A) pRNA and (B) pRNA-3WJ motif. Three domains of pRNA including right-/left-hand loop, foot and central 3WJ provide three different strategies for nanoparticle construction. 3WJ is composed of three RNA oligomers in black (a_3WJ), red (b_3WJ) and blue (c_3WJ). Helical segments are represented as H1, H2 and H3. (C) AFM image of the extended pRNA-3WJ nanoparticles. (D) Sequence of re-engineered pRNA-X motif. The nucleotides in red indicate new sequences that were added to the original pRNA sequences in black. The core is composed of four RNA oligos (denoted a, b, c and d). Helical segments are represented as H1, H2, H3 and H4. Figure adapted and reproduced with permission from: (A) Ref. [14] © 2013 RNA Society; (B) Ref. [9] © 2011 Nature Publishing Group; (C) Ref. [18] © 2015 American Chemical Society; (D) Ref [48] © 2012 Elsevier.

The size, shape, and surface characteristics of nanoparticles play a key role in their biodistribution in vivo [61, 62]. For systematic parallel screening of a myriad of nanoparticle properties, challenges remain in the rapid, precise, and reproducible synthesis of nanoparticle libraries with tunable physicochemical properties and narrow size distribution. RNA nanoparticles provide an emerging robust platform for large-scale preparation of nanoparticles with tailored and controllable physicochemical characteristics, which eventually facilitates high-throughput screening in drug discovery as well as in vivo drug evaluation.

RNA nanoparticles of different shapes

The shape of nanoparticles has been shown to dictate the interaction of nanoparticles with cell membranes and circulating serum proteins [63, 64]. Unlike spherical nanoparticles, oblate-shaped nanoparticles possessing discoidal geometries are more prone to marginate to vessel walls and establish interactions with endothelial cells in blood vessels [65, 66]. The pRNA molecule has been extensively developed as a building block to form multimeric nanoparticles of different shapes by sequence engineering. This is well exemplified by the higher-ordered nanostructures of pRNA dimer up to heptamer assembled via hand-in-hand or foot-to-foot interactions [50, 60]. By re-engineering the pRNA-3WJ motif and introducing another branch, an X-shaped four-way junction was designed as tetravalent nanocarriers (Fig. 1D) [48]. Recent studies demonstrated the angle transition of pRNA-3WJ motifs among RNA triangle, square, and pentagon nanoparticles (Fig. 2) [52]. Changing one RNA strand during polygon assembly induced stretching of the interior angle from 60° to 90° or 108°, resulting in self-assembly of elegant RNA triangles, squares, and pentagons, respectively. The RNA triangle with overhangs was further designed as monomer for assembly of hexamer and honeycomb-shaped structure [51]. Based on the two-dimensional (2D) polygon design, three-dimensional (3D) RNA complexes were developed. Modular design of RNA tetrahedrons [67] and RNA nanoprisms [68] using pRNA-3WJ further demonstrated the feasibility of applying 3WJ for precise control over the shape of RNA nanoparticles (Fig. 2).

Figure 2.

Construction and characterization of multivalent RNA nanoparticles with tunable shape and stoichiometry. Three different strategies were developed for RNA nanoparticle design: (1–5) Loop-loop interactions; (6–10) Foot-to-foot interactions using palindrome sequences; (11–26) Branch grafting based on pRNA-3WJ motif. Figure adapted and reproduced with permission from ref. [14] © 2013 RNA Society, ref. [52] © 2014 Oxford University Press and ref. [71] © 2015 Elsevier.

RNA nanoparticles of different sizes

The effects of size have been extensively studied with spherical NPs and general trends indicate that particles with a diameter in the range of 10 – 100 nm show improved circulation time and tumor accumulation over small molecule counterparts [69]. RNA sequences can be designed by bottom-up self-assembly into precisely-controlled nanostructures. This is exemplified by a modular design of RNA nanosquares with different size ranging from 5 nm to 20 nm [53]. Crystal structure of pRNA-3WJ (Fig. 3A) revealed its planar geometry of three helices (H1, H2, and H3) [70]. By increasing or decreasing the length of the RNA duplex between aligned 3WJ at each corner of the square, nanoparticles of 5, 10, and 20 nm were constructed (Fig. 3B) [53]. For 3D RNA nanoparticles, similar approach by increasing the length of RNA duplex bridging each two adjacent corners can be exploited. As demonstrated by RNA tetrahedrons (Fig. 3C) [67] and nanoprisms (Fig. 3D) [68], the size was well-tuned for favorable in vivo targeting or protection of encapsulated cargos. Inspired by the branched structure of small chemical molecule-based dendrimers, RNA motifs with branched geometry were utilized for supramolecular assembly of multi-layered RNA dendrimer. From Generation 0 (G-0) to Generation 4 (G-4), divergent growth from a core site (G-0) generates globular dendrimers with defined size ranging from 6 nm to 65 nm (Fig. 2) [71].

Figure 3.

Design and characterization of RNA nanoparticles of different sizes. (A) Crystal structure of pRNA-3WJ. (B) AFM images of small (5 nm), medium (10 nm), and large (20 nm) RNA squares derived from pRNA-3WJ. (C) Cryo-EM reconstruction of small (8 nm) and large (17 nm) RNA tetrahedrons derived from pRNA-3WJ. (D) Cryo-EM reconstruction of small (5 nm) and large (10 nm) RNA nanoprisms derived from pRNA-3WJ. Figure adapted and reproduced with permission from: (A) Ref. [70] © 2013 Oxford University Press; (B) Ref. [53] © 2014 American Chemical Society; (C) Ref. [67] © 2016 John Wiley & Sons, Inc. (D) Ref. [68] © 2016 John Wiley & Sons, Inc.

RNA nanoparticles with tunable thermodynamic properties

Thermodynamic stability of nanoparticles is of utmost importance as it is the determining factor of whether the nanoparticle remains intact in vivo after systemic injection. Within nucleic acid nanoparticles, this stability relies primarily on the hydrogen bonding of individual base pairs [72, 73]. It has been shown that RNA motifs display higher thermodynamic stability over their DNA counterparts’ due to added stability from base stacking and tertiary interactions of the RNA nucleotides [55, 56, 74, 75]. This added stability has been demonstrated by the exceptional thermal stability of RNA nanoparticles derived from pRNA-3WJ [9]. The developed RNA nanoparticles remained intact under strong denaturing conditions and at ultra-low concentrations. The novelty behind this pRNA-3WJ structure lies on the finding of its novel energy landscape. Traditionally, macromolecular folding is believed to be driven by enthalpy, the total heat content of a system. Our recent report suggests that the assembly of the 3WJ is governed more by entropy rather than by enthalpy. Entropy is the level of disorder within a system as the randomness between molecules. It was found that the three RNA fragments assembled into the 3WJ with extraordinary speed and affinity via a rapid two-step reaction mechanism, 3WJ_b + 3WJ_c ↔ 3WJ_bc + 3WJ_a ↔ 3WJ_abc. The first step of reaction between 3WJ_b and 3WJ_c is highly dynamic since these two fragments only contain 8 nt for complementation. In this first step enthalpy plays a key role. Immediately following the first step, a second step occurs at a very high association rate, resulting in a rapid formation of the 3WJ seemingly a one-step reaction with a remarkably low Gibbs free energy (ΔG°) of -28 kcal/mol, and a high melting temperature (T_M) of 59.3°C [55]. In the second step, formation of the 3WJ_bc complex generated a dimer with a fixed angle and two protruding helices, presenting an increased 17 nt for binding of 3WJ_a. The addition of the 3WJ_a strand results in locking the unstable 3WJ_bc complex into a highly stable 3WJ. Consequently, the resulting pRNA-3WJ is more stable than any of the dimer species as shown in the much more rapid association rates and slowest dissociation rate constant [55]. When applying the pRNA-3WJ motif in higher-ordered nanoparticles, diverse thermodynamic properties were observed. For example, the T_m of RNA squares increased corresponding to an increase in size. To adjust the thermo-stability of RNA nanoparticles, the substitution of nucleotide (e.g., DNA, 2’-fluorine RNA) in the core strand leads to variable T_m, as evidenced by the RNA squares with varying core strand identities creating hybrid base pairings (Fig. 4A) [53]. It has been shown that chemical modifications on the ribose (e.g., 2’-F, 2’-OMe, locked nucleic acid), or phosphate backbone (e.g., phosphorothioate linkage) improve the stability of RNA constructs enzymatically or thermodynamically. Among these modifications, LNA shows the strongest potential in enhancing the thermodynamic stability of RNA nanoparticles as dictated by a significantly increased T_m. [76]

Figure 4.

RNA nanoparticles with tunable (A) thermal stability, (B) chemical stability and (C) mechanical stability. Figure adapted and reproduced with permission from: (A) Ref. [53] © 2014 American Chemical Society; (B) Ref. [53] © 2014 American Chemical Society; (C) Ref. [82] © AAAS.

RNA nanoparticles with tunable chemical stabilities

The major difference between DNA and RNA is the 2’ location on the ribose, an -OH for RNA and an –H for DNA. The susceptibility of native RNA to nuclease degradation hindered its in vivo applications. To address this issue, chemical modifications to RNA such as 2’-fluorine (2’-F) have shown improved resistance to degradation caused by cellular nucleases [77]. 2’-F pyramidine (C and U) modified RNA drastically enhances chemical and enzymatic stability, rendering a longer half-life of nanoparticles under physiological conditions (Fig. 4B). By changing the ratio of 2’-F modified RNA present in nanoparticles, different stabilities or susceptibilities to RNases were achieved, potentially allowing for temporally-controlled degradation of RNA nanostructures and thus cargo release [53].

RNA nanoparticles with controllable mechanical properties

Unlike mechanically stable proteins [78], nucleic acid structures generally exhibit much lower mechanic stability due in part to lack of long range interactions in their tertiary structure [79–81]. Interestingly, pRNA-3WJ showed exceptional mechanical anisotropy withstanding forces up to 220 ± 78 pN along the H1–H3 portal axis upon Mg²⁺ binding while low mechanical anisotropy was observed in the absence of Mg²⁺. This reversibly modulation of mechanical stability makes pRNA-3WJ a novel structure for construction of biomaterials with controllable mechanical resistance (Fig. 4C) [82].

The elasticity of nanoparticles has recently been viewed as a viable parameter to impact the cellular uptake and intracellular delivery [83]. Elastic deformation of soft nanoparticles significantly facilitates uptake during phagocytosis while clathrin-mediated mechanism dominates the internalization of stiff nanoparticles [23, 84]. RNA nanoparticles were demonstrated to be of desirable elasticity (unpublished data), rendering them favorable for deformation in the process of overcoming series of biological barriers.

RNA nanoparticles with variable immunomodulation effects

The immune system plays a critical role in defending organisms from foreign matters and keeping homeostasis in human body. Inappropriate immune response or hypoimmunity can lead to adverse side effects. Controlling immune response to RNA nanoparticles is critical for developing safe and effective RNA therapeutics. Recent studies suggest that the immune response elicited by RNA nanoparticles is highly dependent on RNA sequence, chemical modifications, size, and shape. The pRNA nanoparticles can be designed to reach two opposite effects: completely non-immunogenic [19, 24, 52] to serve as vehicles for the delivery of therapeutics for disease treatment, or strongly immunogenic leading to induction of TNF-α, IL-6 and other cytokines [52], thus serving as potent vaccine adjuvant or reagents for cancer immunotherapy [85].

Achieving specific targeting of tumors while avoiding entrapment in healthy organs

Specific targeting strategies

Most of the nanoplatforms for drug delivery adopt a “passive” targeting approach relying on EPR effects, which suffers from its random nature and lack of control [86]. In addition, due to tumor heterogeneities, not all cancers exhibit EPR effects, thus losing drug efficacy in some tumors [87]. Clinical data remains unclear on whether the well-documented EPR effect in small animal models also exists in humans [88]. Nonetheless, the major challenge in cancer research remains the non-specific accumulation of therapeutic nanoparticles in healthy vital organs, i.e. liver, lungs, kidneys, and spleen. This low specificity reduces the fraction of nanoparticles that reach tumors, while increasing the toxicity and side effects [62, 89]. Controlled “active” targeting is desirable for successful treatment strategies to effectively block tumor progression and prevent metastasis.

Aptamers refer to single-stranded DNA or RNA molecules selected from an in vitro evolution method [90]. These nucleic acids typically form specific 3D structure with high-affinity to specific cell surface markers via three-dimensional interactions including hydrophobic and electrostatic interactions, hydrogen bonding, and van der Waals forces [91]. With the development of in vitro evolutionary methods (termed SELEX) for the discovery of oligonucleotides that bind to protein targets [92], tens of RNA aptamers have been identified for various cell surface receptors (e.g., EGFR [93], PSMA [94], HER2 [95], EpCAM [96], CD4 [97]). After binding to the receptor, the aptamers can then internalize into the cells via receptor-mediated endocytosis [98]. However, most aptamers themselves are susceptible to renal filtration due to a lower molecular mass compared to the cutoff molecular mass of renal glomerulus [99]. Introducing RNA aptamers to RNA nanoparticle complex increases the circulation time while lowering the off-target effect of RNA nanoparticles. To construct RNA nanoparticles harboring aptamer, functional sequences are fused into core strands without affecting their original folding [17]. For example, fusion of an EGFR-targeting aptamer (EGFRapt) with one of the 3WJ strands produces thermodynamically stable 3WJ-EGFRapt chimera structure with high binding affinity to EGFR overexpression cancer cells [18]. Similarly, PSMA aptamer was incorporated into pRNA-3WJ through bottom-up construction. The PSMA aptamer-tethered pRNA-3WJ nanoparticles showed specific targeting to the LNCaP xenograft tumor compared to their counterpart nanoparticles without PSMA aptamer [20].

As an alternative, chemical ligands can be incorporated as targeting moieties. As a common tumor-enriched antigen, folate receptor (FR) has been identified as a tumor maker, especially in epithelial carcinomas. Folate, the ligand of FR, binds to its receptor with high affinity [100]. In early drug delivery studies, folate was conjugated to cytotoxic agents such as chemical drug and protein toxins [101]. The folate-tethered macromolecules show enhanced delivery to folate-expressing cancer cells in vitro in almost all situations tested. However, folate-mediated macromolecular targeting in vivo has yielded mixed results and is largely due to the non-specific entry of macromolecules into healthy cells [102]. RNA itself, as a polyanionic polymer, disallows non-specific binding to negatively-charged cell membrane [103]. Functionalization of folate or other targeting molecules on RNA nanoparticles significantly enhances the specific recognition of nanoparticles to target cells [14, 19, 21, 48, 49].

Favorable biodistribution of RNA nanoparticles in vivo for efficacious treatment

In vitro targeting to cancer cells has been achieved by many nanoparticles; however, site-specific delivery of therapeutics in vivo faces formidable challenges. Upon intravenous administration, drug-containing nanoparticles encounter a series of biological barriers. Under physiological conditions, nanoparticles undergo rapid opsonization and subsequent sequestration by resident macrophage of the mononuclear phagocyte system (MPS), resulting in significant accumulation of nanoparticles in healthy organs such as the liver and spleen [104, 105]. To overcome these obstacles, RNA nanoparticles were exploited to reach specific target sites and bypass the normal organ accumulation and renal clearance (Table 1). As examples, an RNA aptamer targeting epidermal growth factor receptor (EGFR) was anchored on pRNA-3WJ nanoparticles (Fig. 7A) for specific targeting to triple negative breast cancer (TNBC) (Fig. 5A). Specific targeting and retention of RNA nanoparticles in tumors was evidenced by the histological assay of tumor frozen cross sections using fluorescence confocal microscopy (Fig. 7B). Compared to the control group, treatment with pRNA-3WJ-EGFR displayed remarkable accumulation at tumor site without non-specific accumulation in healthy organs [18]. Similarly, 3D RNA tetrahedron nanoparticles decorated with EGFR aptamer showed tumor-specific enrichment in orthotopic MDA-MB-231 tumor-bearing mice after systemic administration [67]. Another successful aptamer-guided in vivo targeting is exemplified by pRNA-3WJ nanoparticle harboring anti-prostate-specific membrane antigen (PSMA) RNA aptamer (Fig. 5D) [20]. An alternative strategy to improve the targeted delivery of nanomedicine into solid tumors is folate conjugation on pRNA-3WJ nanoparticles. The pRNA-3WJ-folate conjugates have been successfully exploited in various FR overexpressed cancer types including glioblastoma (Fig. 5B) [21], gastric cancer (Fig. 5C) [19], colorectal cancer (Fig. 5E) [49], head & neck cancer (Fig. 5F) [14]. To study the in vivo biodistribution of RNA nanoparticles over the course of time, pRNA-X nanoparticles harboring folate and fluorophore were systemically injected into athymic nude mice bearing KB cells xenografts. At different time points, whole-body imaging showed the significant accumulation of RNA nanoparticles in tumor within 4 hours (Fig. 6A). The organ imaging at 8-hour time point indicated the specific localization of pRNA-X nanoparticles in tumor without entrapment in the normal organs (Fig. 6B) [48]. Together, several RNA nanoparticle platforms have demonstrated the superior ability of RNA nanoparticles to lead to selective targeting of diseased tissues over other nanotechnology platforms. This targeting overcomes a major hurdle that is currently limiting FDA approved therapeutics and will advance the pharmaceutical field in the future.

Table 1.

RNA nanoparticles for successful in vivo specific targeting

Strategy	Related marker	RNA nanoparticle	Model	Ref
Aptamer	EGFR	pRNA-3WJ; RNA tetrahedron	Breast cancer	[18,67]
	PSMA	pRNA-3WJ	Prostate cancer	[20]
Folic acid	FR	pRNA-X	KB xenograft	[48]
	FR	pRNA-3WJ	Colorectal cancer; Glioblastoma; Gastric cancer; Head & neck cancer	[49] [21] [19] [14]
Ocular Delivery		pRNA-X	Retina cells	[110]
		pRNA	Cornea	[110]

FR, folate receptor; pRNA, packaging RNA; 3WJ, three-way junction

Figure 7.

Evaluation of the therapeutic effects of 3WJ-EGFRapt/anti-miRNA21. (A) Fluorescence images showing specific targeting and retention in TNBC tumors 8 h post-injection. (B) Histological assay of breast tumor frozen cross sections showing binding and internalization. Blue: nuclei; red: RNA nanoparticle. (C) Tumor growth curve over the course of 5 injections. (*P<0.05, **P<0.01, error bars indicate SEM). (D) Tumor inhibition over the course of 5 injections. (E) Western blot and (F) Real-time PCR showing the down-regulation of miRNA21 after treatment, resulting in up-regulation of two target genes PTEN and PDCD4. RQ: relative quantification. Figures adapted with permission from ref [18] © 2015 American Chemical Society.

Figure 5.

Specific targeting of RNA nanoparticles to various xenografts, metastasis, and ocular tissues. Whole-body and organ imaging of (A) Brain cancer; (B) Breast cancer; (C) Gastric cancer; (D) Prostate cancer; (E) Colorectal cancer; (F) Head & neck cancer; (G) CRC metastasis in the lung, liver, lymph node and bones. Green: GFP-cancer cells; blue: DAPI; red: RNA nanoparticles. (H) Ocular fluorescence imaging of the eye after subconjunctival injection of pRNA-X. Figure adapted and reproduced with permission from: (A) Ref. [21] © 2015 Impact Journals; (B) Ref. [18] © 2015 American Chemical Society; (C) Ref. [19] © 2015 Macmillan Publishers Limited, part of Springer Nature; (D) Ref. [20] © 2016 Elsevier; (E) Ref. [49] © 2015 American Chemical Society; (F) Ref. [14] © 2013 RNA Society; (G) Ref. [49] © 2015 American Chemical Society; (H) Ref. [110] © 2013 Springer US.

Figure 6.

Specific targeting and accumulation of pRNA-X nanoparticles to KB cells xenograft. (A) Whole-body imaging at different time points. (B) Internal organ imaging. Figure adapted and reproduced with permission from ref [48] © 2012 Elsevier.

Despite the favorable distribution of RNA nanoparticles in tumor xenograft model, accumulation of drug-containing nanoparticles in metastases is especially important. As the most lethal aspect of cancer, metastasis is one of the greatest challenges in cancer treatment today due to their small size, high multiplicity, and dispersion to diverse organ environments [106]. The major sites of metastasis include lungs, liver, and lymph nodes [107]. To date, an effective and safe system capable of exclusively targeting metastatic cancers is urgently needed. Rychahou et al reported the specific delivery of folate-conjugated pRNA nanoparticles into colorectal cancer metastases (Fig. 5G). After systemic injection, the RNA nanoparticles bind to CRC liver, lung, and lymph node metastases without accumulation in normal liver, lung, and other healthy organs [49].

To validate the targeting, the therapy effects of RNA nanoparticles harboring therapeutic agents were evaluated in tumor-bearing mice. EGFRapt-guided delivery of anti-miR21 displayed efficacious inhibition of tumor growth in TNBC mouse model [19]. As shown in the tumor growth curve (Fig. 7C) and luminescence signal (Fig. 7D), treatment of 5 doses of 3WJ-EGFRapt/anti-miRNA21 resulted in sustained inhibition of tumor growth compared to control group. The knockdown effect of miRNA-21 was also revealed at both mRNA (Fig. 7F) and protein levels (Fig. 7E) with increased expression of downstream PTEN and PDCD4. Another study using PSMA aptamer for specific delivery of anti-miRNA17 and anti-miRNA21 also showed high efficient inhibition of prostate cancer [20]. Chemical ligand such as folate has proved to successfully deliver siRNA to glioblastoma, leading to significant gene silencing within the luciferase gene expressing brain tumors [21].

The favorable in vivo pharmacokinetics of RNA nanoparticles also yield potential benefits for ocular disease treatment. Current ocular delivery of therapeutics such as oligonucleotides to posterior segment of the eye requires repeated intravitreal injections, which involves severe side effects such as intraocular bleeding, infection and retinal detachment [108]. Specific delivery of therapeutics to the posterior segment of the eye is desirable yet challenging [109]. Subconjunctival injection of pRNA-3WJ, pRNA-X nanoparticles, and double-stranded RNA (dsRNA) showed different PK profiles. Cell internalization was observed in the cornea with pRNA-3WJ and pRNA-X nanoparticles. However, only the pRNA-X nanoparticles could enter the retina (Fig. 5H) [110]. These pharmacokinetic studies provided useful information on the distribution of drugs in the eye and in the development of novel drug delivery methods.

In general, the favorable biodistribution of RNA nanoparticles is due to several reasons. The negative charge of RNA minimizes nonspecific binding to negatively charged cell membrane, thus reducing the toxicity associated with normal cells. RNA nanoparticles are homogenous with defined structure, size, and stoichiometry and thus can avoid nonspecific side effects. RNA nanoparticles are 10–50 nm in size and sufficient to harbor siRNAs, ribozymes, miRNAs, and RNA aptamers [111]. They are large enough to decrease rapid renal excretion, yet small enough to enter cells by receptor-mediated endocytosis, while avoiding entrapment by liver Kupffer cells and lung/spleen macrophages. The elastic nature and branched ratchet shape of our RNA nanoparticles facilitate tumor penetration and increases EPR effects. This is particularly useful for overcoming mechanical barriers, disorganized vascularization, and highly immunosuppressive tumor microenvironments. A summary of the advantageous of RNA nanoparticles for in vivo applications is listed in Box 1.

Box 1. Advantages of RNA nanoparticles for in vivo applications.

• Controllable size, shape and surface characteristics

• Thermodynamically stable to avoid in vivo disociation

• Three-dimensional nanoparticle with cage-like structure to prevent cargo from digestion and diffusion

• Negatively-charged phosphate backbone disallows non-specific binding to cell membrane

• Biocompatibility and no toxicity

• Programmable with targeting ligands, therapeutic agents and imaging module

The effect of conjugated chemicals on the in vivo biodistribution of RNA nanoparticles

Nanoparticle clearance from the reticuloendothelial system (RES) depends not only on its size but also on the surface modification and can vary significantly among the different properties of chemicals conjugated to nanoparticles [61, 112]. Tremendous efforts have been made to increase water solubility of nanoparticles for longer circulation such as polyethylene glycol (PEG) grafting on nanoparticles [113, 114]. Studies have shown that the nanoparticle size and surface composition (e.g., hydrophobicity) are very important parameters in defining the composition of the formed protein corona. More hydrophobic nanoparticles bound more significant amounts of protein and the association and dissociation rates of bound proteins were clearly dependent on the hydrophobicity of the particles [115, 116]. RNA, as a natural water-soluble polymer, has been engineered as multifunctional nanoparticles by decorating fluorophore, targeting ligands, and therapeutic agents. However, little is known about the effects of conjugating hydrophobic fluorophores on plasma protein binding as well as organ accumulation. Jasinski et al investigated the changes in vital organ accumulation patterns resulting from surface hydrophobicity variation. Three fluorophores of different hydrophobicities were conjugated to the 3WJ RNA nanoparticle to serve as model chemicals: Cyanine5.5 (Cy5.5); Sulfonated-Cyanine 5.5 (SulfoCy5.5); and AlexaFluor700. RNA nanoparticles with conjugated fluorophore (3WJ-fluor) exhibited decreased fluorescent signal in vital organs, especially in lung (Lu) and liver (Lv), when compared to that of fluorophore alone. This is strong indication of less accumulation in vital organ for 3WJ-fluor nanoparticles [117].

As shown here, the biological impacts of RNA nanoparticles are closely related to the modified chemicals following systemic administration. While changes in RNA nanoparticle protein binding might be difficult to predict, understanding the effects of conjugating hydrophobic drugs on its solubility, and in turn in vivo biodistribution can help researchers develop RNA-based therapeutics in future clinical settings.

Excretion and clearance of RNA nanoparticles

As growing evidence has demonstrated the favorable biodistribution of rationally designed RNA nanoparticles in vivo, a basic understanding of how RNA nanoparticles are eliminated is important for their future clinical applications. In general, nanoparticles undergo excretion and clearance by renal filtration, with excretion into the urine, or hepatobiliary processing, with excretion into the bile [118, 119]. Based on the urine assay of 3WJ nanoparticles, it’s suspected that kidney filtration into the urine is the major excretion pathway for RNA 3WJ nanoparticles [76]. Though in vitro serum stability assays and tumor pharmacokinetic models have characterized the biological and distribution effects of RNA nanoparticles, more accurate quantitative assays are needed to streamline in vivo studies.

Conditional endosomal escape of RNA nanoparticles

Specific targeting and favorable in vivo biodistribution of RNA nanoparticles ensure the delivery of therapeutic agents to the target cancer cells; however, their intracellular bioavailability is also critical for efficacious therapy. Nanoparticles are typically taken up into cells via endocytic pathway where they are entrapped in endosomes and are then subsequently trafficked into lysosomal compartments. The acidic environment with specific enzymes in the lysosome leads to significant degradation of the therapeutics [120]. The pH value of endosome especially lysosome is low, but RNA is relatively resistant to low pH, a property different from that of DNA which is resistant to high pH but is subject to depurination at low pH. Recent studies on nanoparticle intracellular trafficking indicate that the siRNA delivery efficiency is significantly hindered by insufficient endosomal escape [121]. Hence, a rate-limiting yet challenging barrier in achieving an effective therapy is to facilitate the endosomal escape and ensure cytosolic delivery of the therapeutics.

Despite the success of RNA nanoparticle in cancer therapy, little is known about its internalization pathway and subsequent intracellular trafficking. Ample in vivo cancer treatment outcomes indirectly suggested successful endosomal escape of RNA nanoparticles to exert therapeutic effects. For example, pRNA-3WJ has been exploited for efficient delivery of anti-miRNA with more significant gene regulation effects compared to that induced by siRNA [18].

Different strategies have been developed to facilitate release of nanoparticles from endosome before lysosomal degradation [122]. General mechanisms are proposed such as pore formation in the endosomal membrane, “proton sponge” effect of protonable groups and fusion into the lipid bilayer of the endosome [123, 124]. Of note, the protonable molecules induce an extensive inflow of ions and water into the endosome, leading to osmotic swelling and subsequently rupture of the endosomal membrane. Imidazole and other pH sensitive groups (e.g., hydrazine, maleic amides, amino esters) are typically incorporated into the nanoparticles for an enhanced pH buffering capacity [125–127]. As many conjugation methods for end-labeling of RNA have been established, conjugation of endosome disrupting compounds to RNA nanoparticles poses remarkable impacts in surmounting endosomal barriers.

Challenges and limitations of RNA nanotechnology for biomedical applications

Although great progress has been achieved by applying RNA nanotechnology for biomedical applications, the potency of RNA nanoparticles from a translational perspective is crucially limited by their inherent physicochemical characteristics [18, 67, 68].

One of the major bottlenecks for RNA nanotechnology in therapeutic applications is the large-scale production and high cost of the nanoparticle construction. RNA oligonucleotides prepared by solid phase synthesis are in maximum 80 nt long, preventing nanoparticle designers from using longer sequence in the modular design of RNA nanostructure. The high cost of RNA synthesis also limits the industry scale production of RNA nanoparticles though efforts in improving chemical synthesis have been made [128, 129].To improve the recovery of RNA nanoparticles, purification methods such as preparative ultracentrifugation have been developed to overcome the low yield of gel electrophoresis and HPLC purification [54]. Referring to the progressively reduced cost of DNA oligosynthesis over the years [130], it is expected that the cost of RNA production will gradually decrease with the improvements in chemical synthesis efficiency and production facilities developed in industries [128, 129]. Production of RNA by biological approaches such as bacterial fermentation in combination with intracellular assembly of RNA nanoparticles has also been proposed [10].

The active targeting strategy deployed on RNA nanotechnology is successful yet challenging. Given the heterogeneous nature of cancer, specific targeting ligand is required for each cancer type [131, 132]. However, the number of available RNA aptamers and ligands is limited and insufficient. Rapid screening methods derived from SELEX have shown considerable promise to keep up with the emergence of RNA-based therapeutics [111]. Computational methods such as structural modeling and prediction have shown to facilitate the experimental screening process of aptamer [133–135].

With the advancement in material science and engineering, nanoparticle system offers a promising opportunity for targeted cancer therapy. However, a recent report showed that only 0.7% (median) of the administered nanoparticle dose accumulates in tumor based on the analysis of publications in “nanoparticle delivery” over last 10 years [136]. This finding brought up concerns for all nanoparticle-based specific targeting and drug delivery systems. Current findings on RNA nanoparticle in vivo behaviors such as low entrapment in healthy vital organs, rich accumulation in tumors and high efficiency in inhibiting cancer growth have provided evidence that a significant amount of administered RNA nanoparticles accumulate in tumors. However, as RNA nanotechnology for cancer treatment is merely at starting point, multi-parametric studies and quantification analysis are needed for better understanding and interpretation of the in vivo behavior of RNA nanoparticles with different synthetic and biological identities. With more fundamental studies on the pharmacokinetics of RNA nanoparticles, optimized design on their physicochemical properties including size, shape, and chemical modification will be guided for improved specific targeting and delivery efficiency.

Conclusions

Nanoparticles have evolved considerably in preclinical and clinical studies for cancer targeting and therapy in the past two decades; it is critical to overcome the biological barriers that hinder the in vivo targeting and intracellular delivery of nanoparticles. Though present-day nanoparticle-based therapy is extending beyond the confines of convention and transitioning toward more rational design, there is the growing realization that the distinct obstacles are by no means insurmountable. RNA nanotechnology-based therapy shed new light on innovative design of therapeutics for clinic translation.

As highlighted here, two major active targeting strategies implemented on pRNA-3WJ based RNA nanoparticles to specific cancer were demonstrated in different tumor models. Given the highly heterogeneous and continuously evolving nature of the tumor, developing tumor-specific RNA nanoparticles benefits the optimized delivery in personalized medicine. In virtue of the nature of RNA as building block in nanoparticle design, physicochemical properties of RNA complex are also tunable. Understanding the effects of RNA nanoparticle size, shape, and surface chemistry on its biodistribution in biological systems will assist in rational design of entities that are capable of sequential negotiation of biological obstacles for efficacious, site-specific delivery. Increasing evidence has revealed the correlation of physicochemical properties of RNA nanoparticles with their in vivo PK profiles. However, an in-depth understanding of their intracellular trafficking as well as endocytic pathway is needed. Ultimately, this may not only lead to successful translation of novel RNA-based therapeutics, but will also guide the design of precision nanomedicine towards treatment of diseases.

Highlights.

• The novelty of pRNA derived from phi29 DNA packaging motor in RNA nanotechnology is described.

• Specific targeting of tumors using RNA nanoparticles by different strategies is summarized.

• Effects of physicochemical properties of RNA nanoparticle on biodistribution are discussed.