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Cancer Letters 414 (2018) 57e70

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Cancer Letters

journal homepage: www.elsevier .com/locate/canlet

Mini-review

Favorable biodistribution, specific targeting and conditionalendosomal escape of RNA nanoparticles in cancer therapy

Congcong Xu a, b, c, d, 1, Farzin Haque e, 1, Daniel L. Jasinski a, b, c, d, Daniel W. Binzel a, b, c, d,Dan Shu a, b, c, d, Peixuan Guo a, b, c, d, *

a Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USAb College of Medicine, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USAc Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USAd Center for RNA Nanobiotechnology and Nanomedicine, The Ohio State University, Columbus, OH, USAe Nanobio Delivery Pharmaceutical Co. Ltd., Columbus, OH, USA

a r t i c l e i n f o

Article history:Received 23 July 2017Received in revised form14 September 2017Accepted 25 September 2017

Keywords:RNA nanotechnologyphi29 motor pRNApRNA-3WJ motifNanobiotechnologyBiodistributionCancer therapy

* Corresponding author. Peixuan Guo, Sylvan G. Frmaceutics and Drug Delivery, The Ohio State UniversTower (BRT), 460 W 12th Ave. Columbus, OH 43210,

E-mail address: guo.1091@osu.edu (P. Guo).1 Co-first author.

https://doi.org/10.1016/j.canlet.2017.09.0430304-3835/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

The past decades have witnessed the successful transition of several nanotechnology platforms into theclinical trials. However, specific delivery of therapeutics to tumors is hindered by several barriersincluding cancer recognition and tissue penetration, particle heterogeneity and aggregation, and unfa-vorable pharmacokinetic profiles such as fast clearance and organ accumulation. With the advent of RNAnanotechnology, a series of RNA nanoparticles have been successfully constructed to overcome many ofthe aforementioned challenges for in vivo cancer targeting with favorable biodistribution profiles.Compared to other nanodelivery platforms, the physiochemical properties of RNA nanoparticles can betuned 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 onthe in vivo fate of RNA nanoparticles. Rationally designed RNA nanoparticles with defined stoichiometryand high homogeneity have been demonstrated to specifically target tumor cells while avoiding accu-mulation in healthy vital organs after systemic injection. RNA nanoparticles were proven to delivertherapeutics such as siRNA and anti-miRNA to block tumor growth in several animal models. Althoughthe release of anti-miRNA from the RNA nanoparticles has achieved high efficiency of tumor regressionin multiple animal models, the efficiency of endosomal escape for siRNA delivery needs furtherimprovement. This review focuses on the advances and perspectives of this promising RNA nanotech-nology platform for cancer targeting and therapy.

© 2017 Elsevier B.V. All rights reserved.

Introduction

Efficacious responses in patients across a wide range of diseasesrely heavily on the bioavailability and delivery of drugs specificallyat sites of interest. Cancer represents the best example of a diseasewhere potent yet toxic chemotherapeutics can make the differencebetween positive outcomes and severe side-effects [1]. Despitesignificant financial investment, present-day formulations result insignificant accumulations in healthy vital organs and target tumorswith low specificity. Addressing such issues, nanoparticle-based

ank Endowed Chair in Phar-ity, 912 Biomedical ResearchUSA.

drug delivery is emerging as a powerful tool to alter the pharma-cokinetics and biodistribution of chemotherapeutic and RNAi baseddrugs administered systemically [2]. By shifting the balance be-tween off- and on-target accumulation, several nanomedicinessuch as Doxil™ (liposomal doxorubicin) and Abraxane™ (pacli-taxel-containing albumin nanoparticles) have gained success in theclinic [3]. Even so, most current drug delivery platforms face limitedsite-specific bioavailability due to a series of roadblocks associatedwith formulation challenges (particle heterogeneity, particle ag-gregation, particle dissociation, high production costs, and unstablethermodynamic and chemical properties); unfavorable bio-distribution and pharmacological profiles; and difficulty to over-come biological barriers surrounding tumors [4,5]. In addition, lackof controlled-release slows down the clinical translation. Thecomplex composition of nanocarriers with diverse functionalmodules (inorganic/organic nanoscaffolds, RNA/protein antibodies

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combination, chemical drugs/antibodies complexing) also delaysregulatory approval of these nanoparticles [6,7].

With the advancement of RNA nanotechnology field over thepast decade, RNA-based nanoparticles have shown great potentialfor delivering therapeutics in vivo with favorable pharmacologicalprofiles [8e11]. RNA, as a naturally-occurring polymer, is advan-tageous as a building block for bottom-up assembly of nano-particles with defined size, shape, and stoichiometry, composedpurely of RNA nucleotides [12]. RNA exhibits both canonicalWatson-Crick (A-U, G-C) and noncanonical (such as G-U) basepairing [13] and this property along with base stacking and tertiaryinteractions enable the RNA to fold into complex structural con-formations [28]. Several RNA nanoparticles have been constructedwith tunable chemical and thermodynamic properties suitable forin vivo drug delivery [14]. More recently, the three-way junction(3WJ) motif of packaging RNA (pRNA) molecules derived from thephi29 DNA packaging motor have shown great potential in devel-oping RNA nanoparticles for cancer targeting and therapy [9]. The3WJ nanoparticles can be functionalized with varieties of imaging(fluorophores [15] or fluorogenic aptamers [16,17]), targeting (RNAaptamers 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 orfunction of the incorporated modules [17]. Upon systemic injectionin tumor-bearing mice, the pRNA-3WJ based RNA nanoparticlesspecifically accumulate at the tumor site. The elastic nature andbranched ratchet shape of RNA nanoparticles facilitates tumorpenetration and increases EPR (enhanced permeability and reten-tion) effects [23]. This is particularly useful for overcoming me-chanical barriers, disorganized vascularization, and highlyimmunosuppressive tumor microenvironments. Incorporatingtargeting ligands allows RNA nanoparticles to bind cell surface re-ceptors and enter cells through receptor mediated endocytosis andin the process, deliver therapeutics to the cells. Little or no accu-mulation of RNA nanoparticles is observed in vital organs. Thenegatively charged backbone of RNA minimizes nonspecific bind-ing to the negatively charged cell membrane, thus reducing toxicityand side effects. Moreover, the pRNA-based nanoparticles displayfavorable pharmacological profiles, are non-toxic, and induceextremely low interferon or cytokine production in mice [19,24].These promising results indicate that RNA nanotechnology-baseddelivery platforms can potentially solve many of the current chal-lenges that other nanoparticles typically face in cancer therapy.Enhanced therapeutic effects of anti-cancer agents are anticipatedwhen using these platforms, while specific delivery to cancer cellswill reduce the required dose and associated side effects. The easeof incorporation of RNAi therapeutics will also make the platformideal to treat cancers by targeting undruggable mutations, tacklingdrug-resistance, and preventing cancer recurrence. From a trans-lational perspective, RNA nanoparticles are biocompatible, well-characterized, and the production process is relatively easy,scalable, reproducible, and cost effective. Thus, the translation ofRNA nanotechnology to a clinical setting is highly likely. Here weprovide a brief review focusing on the defining aspects of RNAnanoparticles' designs and tuning the physiochemical properties ofRNA 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 forconstructing RNA nanoparticles with various shapes, sizes, andstoichiometry (reviewed in [8,10e12,25]). Briefly, methods include:

(1) Loop-loop or hand-in-hand interactions (complementary se-quences form interlocking loops) [14,26,27]; (2) Foot-to-foot in-teractions (palindrome sequence mediated self-assembly) [14,26];(3) Rational design using RNA motifs, such as kissing loops, dove-tails, pseudoknots, kink turns, and multi-way junctions [28e30];(4) RNA architectonics (rational design of 3D structures fromknown structures of RNA) [30e35] from databases such as theNucleic Acid Database (NAD) [36] and RNAjunction database [37];(5) Computational approaches (computer assisted de novo 3Ddesign) using software programs, such as Nanotiler [38], Assemble2[39], RNA2D3D [40], INFO-RNA [41], and NUPACK [42]; (6) Co-transcriptional assembly (generation of RNA nanoparticles from aDNA template during in vitro transcription) [43e45]; (7) RollingCircle Transcription (generation of concatemeric RNA sequencesfrom a circular DNA template during in vitro transcription) [46,47];and (8) RNA origami (construction of complex shapes from a singlestranded 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 (<80nucleotides) can be synthesized chemically using an oligo synthe-sizer based on solid phase phosphoramidite chemistry. Modifiednucleotides (such as, 20-F, 20-OMe, LNA, etc.) can be incorporated atthe desired location in the sequence. Longer RNA strands (>80nucleotides) need to be transcribed from a DNA template using T7RNA polymerase. A limited number of modified nucleotides can beincorporated using a mutant RNA polymerase.

Currently the chemical synthesis method is only feasible forshort RNA strands. Synthesis of RNAwith sequences longer than 80nucleotides will result in low yield. By applying RNA nanotech-nology, larger RNA particles can be built via bottom-up assemblyusing smaller RNA fragments.

Functionalization and labeling of RNA nanoparticles

RNA based ligands, such as targeting aptamers; therapeuticsiRNA, miRNA, anti-miRNA, and ribozymes; and regulatory mod-ules, such as, ribozymes can be rationally incorporated into RNAnanoparticle scaffolds simply by sequence fusion[9,14,18,19,21,48e53]. Small molecules, such as, targeting ligandsfolate and galactose; fluorophores, such as, Alexa647; or chemicaldrugs can be conjugated to RNA strands using standard chemicalreactions (reviewed in [11]). These include: (1) NHS chemistry viacoupling of an activated carboxylic group with a primary aminegroup on an oligo; (2) Bioorthogonal click chemistry between theazide group on the ligand and the alkyne group on an oligo; (3)Thioester linkage using a thio-modified oligo and the maleimide ona ligand; (4) EDC (carbodiimide) crosslinking through aminogroups and carboxylic acid groups; and, (5) Coupling of a phos-phoramidite derivative of the labeling group with the oligo duringsolid phase synthesis.

Self-assembly of RNA nanoparticles

Typically, the component strands of RNA nanoparticles aremixed at stoichiometric ratios at room temperature or annealed(heated to 95 �C and slowly cooled to room temperature). Differentfrom bottom-up assembly, RNA nanoparticles can also be assem-bled co-transcriptionally [43e45] or through rolling cycle tran-scription [46,47].

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Purification and characterization of RNA nanoparticles

Individual strands are typically purified by HPLC or denaturingPAGE (polyacrylamide gel electrophoresis). Assembled RNA nano-particles are generally purified by nondenaturing PAGE, HPLC, orultracentrifugation [54]. The RNA nanoparticles are then charac-terized as follows: (1) Asses RNA nanoparticle folding and assemblyusing native PAGE gels [14]; (2) Assess Tm by qPCRwith SYBRGreen,temperature gradient gel or UV absorbance [55]; (3) Assess Kd bycompetition assays using radiolabeled RNA [9] or Surface PlasmonResonance [56]; (4) Evaluate chemical stability by incubating RNAwith RNase or 50% FBS [9,57]; (5) Examine resistance to Urea bydenaturing PAGE [9,50]; (6) Structural characterization by AtomicForce Microscopy (AFM) imaging [14,32,58]; and (7) 2D structureprediction by 'mfold' [59].

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

The first proof-of-concept demonstrating the feasibility of RNAnanotechnology was published in 1998 in Molecular Cell (featuredin Cell) [60]. Dimer, trimer, and hexamer complexes were con-structed by bottom-up assembly using reengineered pRNA

Fig. 1. Sequence of (A) pRNA and (B) pRNA-3WJ motif. Three domains of pRNA including rparticle construction. 3WJ is composed of three RNA oligomers in black (a3WJ), red (b3WJ) anextended pRNA-3WJ nanoparticles. (D) Sequence of re-engineered pRNA-X motif. The nucleoblack. The core is composed of four RNA oligos (denoted a, b, c and d). Helical segments are re(A) Ref. [14] © 2013 RNA Society; (B) Ref. [9] © 2011 Nature Publishing Group; (C) Ref. [18] © 2references to colour in this figure legend, the reader is referred to the web version of this

fragments using hand-in-hand or loop-loop interactions (Fig. 1A). Asecond approach relied on foot-to-foot interactions to connect twopRNA monomers using palindrome sequence mediated self-assembly [14]. The pRNA monomer contains a 3WJ motif in thecentral domain (Fig. 1B). More recently, it was shown that the 3WJmotif can be extracted from the core pRNA sequence and used as ascaffold, which represents a third technique for RNA nanoparticleconstruction [9,19,21,49]. Atomic force microscopy (AFM) charac-terization of extended 3WJ showed the branched structure,allowing for incorporation of multiple modules (Fig. 1C) [18]. Overthe past decade, Guo and co-workers have used these three ap-proaches to design a series of RNA nanoparticles with defined size,shape, and stoichiometry.

The size, shape, and surface characteristics of nanoparticles playa key role in their biodistribution in vivo [61,62]. For systematicparallel screening of amyriad of nanoparticle properties, challengesremain in the rapid, precise, and reproducible synthesis of nano-particle libraries with tunable physicochemical properties andnarrow size distribution. RNA nanoparticles provide an emergingrobust platform for large-scale preparation of nanoparticles withtailored and controllable physicochemical characteristics, thateventually facilitates high-throughput screening in drug discoveryas well as in vivo drug evaluation.

ight-/left-hand loop, foot and central 3WJ provide three different strategies for nano-d blue (c3WJ). Helical segments are represented as H1, H2 and H3. (C) AFM image of thetides in red indicate new sequences that were added to the original pRNA sequences inpresented as H1, H2, H3 and H4. Figure adapted and reproduced with permission from:015 American Chemical Society; (D) Ref [48] © 2012 Elsevier. (For interpretation of the

article.)

Fig. 2. Construction and characterization of multivalent RNA nanoparticles with tunable shape and stoichiometry. Three different strategies were developed for RNA nanoparticledesign: (1e5) Loop-loop interactions; (6e10) Foot-to-foot interactions using palindrome sequences; (11e26) Branch grafting based on the pRNA-3WJ motif. Figure adapted andreproduced with permission from Ref. [14] © 2013 RNA Society, Ref. [52] © 2014 Oxford University Press and Ref. [71] © 2015 Elsevier.

C. Xu et al. / Cancer Letters 414 (2018) 57e7060

C. Xu et al. / Cancer Letters 414 (2018) 57e70 61

RNA nanoparticles of different shapes

The shape of nanoparticles has been shown to dictate theinteraction of nanoparticles with cell membranes and circulatingserum proteins [63,64]. Unlike spherical nanoparticles, oblate-shaped nanoparticles possessing discoidal geometries are moreprone to migrate to vessel walls and establish interactions withendothelial cells in blood vessels [65,66]. The pRNA molecule hasbeen extensively developed as a building block to form multimericnanoparticles of different shapes by sequence engineering. This iswell exemplified by the higher-ordered nanostructures of pRNAdimers up to heptamers assembled via hand-in-hand or foot-to-foot interactions [50,60]. By re-engineering the pRNA-3WJ motifand introducing another branch, an X-shaped four-way junctionwas designed as a tetravalent nanocarrier (Fig. 1D) [48]. Recentstudies demonstrated the angle transition of pRNA-3WJ motifsamong RNA triangle, square, and pentagon nanoparticles (Fig. 2)[52]. Changing one RNA strand during polygon assembly inducedstretching of the interior angle from 60� to 90� or 108�, resulting inself-assembly of elegant RNA triangles, squares, and pentagons,respectively. The RNA trianglewith overhangswas further designedas amonomer for the assembly of hexamer and honeycomb-shapedstructures [51]. Based on the two-dimensional (2D) polygon design,three-dimensional (3D) RNA complexes were developed. Modulardesign of RNA tetrahedrons [67] and RNA nanoprisms [68] usingpRNA-3WJ further demonstrated the feasibility of applying 3WJ forprecise control over the shape of RNA nanoparticles (Fig. 2).

Fig. 3. Design and characterization of RNA nanoparticles of different sizes. (A) Crystal structRNA squares derived from pRNA-3WJ. (C) Cryo-EM reconstruction of small (8 nm) and large ((5 nm) and large (10 nm) RNA nanoprisms derived from pRNA-3WJ. Figure adapted and repr© 2014 American Chemical Society; (C) Ref. [67] © 2016 John Wiley & Sons, Inc. (D) Ref. [68

RNA nanoparticles of different sizes

The effects of size have been extensively studied with sphericalNPs and general trends indicate that particles with a diameter in therange of 10e100 nm show improved circulation time and tumoraccumulation over small molecule counterparts [69]. RNA se-quences can be designedbybottom-up self-assembly intoprecisely-controlled nanostructures. This is exemplified by a modular designof RNAnanosquareswith different sizes ranging from5nm to20nm[53]. The crystal structure of pRNA-3WJ (Fig. 3A) revealed its planargeometry of three helices (H1, H2, and H3) [70]. By increasing ordecreasing the length of the RNA duplex between an aligned 3WJ ateach corner of the square, nanoparticles of 5, 10, and 20 nm wereconstructed (Fig. 3B) [53]. For 3D RNA nanoparticles, a similarapproach by increasing the length of RNA duplex bridging twoadjacent corners can be exploited. As demonstrated by RNA tetra-hedrons (Fig. 3C) [67] and nanoprisms (Fig. 3D) [68], the size waswell-tuned for favorable in vivo targeting or protection of encapsu-lated cargos. Inspired by the branched structure of small chemicalmolecule-based dendrimers, RNA motifs with branched geometrywere utilized for supramolecular assembly of multi-layered RNAdendrimers. From generation 0 (G-0) to generation 4 (G-4), diver-gent growth from a core site (G-0) generates globular dendrimerswith defined size ranging from 6 nm to 65 nm (Fig. 2) [71].

RNA nanoparticles with tunable thermodynamic properties

Thermodynamic stability of nanoparticles is of utmost impor-tance as it is the determining factor of whether the nanoparticle

ure of pRNA-3WJ. (B) AFM images of small (5 nm), medium (10 nm), and large (20 nm)17 nm) RNA tetrahedrons derived from pRNA-3WJ. (D) Cryo-EM reconstruction of smalloduced with permission from: (A) Ref. [70] © 2013 Oxford University Press; (B) Ref. [53]] © 2016 John Wiley & Sons, Inc.

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remains intact in vivo after systemic injection. Within nucleic acidnanoparticles, this stability relies primarily on the hydrogenbonding of individual base pairs [72,73]. It has been shown thatRNA motifs display higher thermodynamic stability over their DNAcounterparts' due to added stability from base stacking and tertiaryinteractions of the RNA nucleotides [55,74,75]. This added stabilityhas been demonstrated by the exceptional thermal stability of RNAnanoparticles derived from the pRNA-3WJ [9]. The developed RNAnanoparticles remained intact under strong denaturing conditionsand at ultra-low concentrations. The novelty behind this pRNA-3WJstructure lies on the finding of its novel energy landscape. Tradi-tionally, macromolecular folding is believed to be driven byenthalpy, the total heat content of a system. Our recent reportsuggests that the assembly of the 3WJ is governed more by entropyrather than by enthalpy [55]. Entropy is the level of disorder withina system as the randomness between molecules. It was found thatthe three RNA fragments assembled into the 3WJ with extraordi-nary speed and affinity via a rapid two-step reaction mechanism,3WJb þ 3WJc 4 3WJbc þ 3WJa 4 3WJabc [56]. The first step of thereaction between 3WJb and 3WJc is highly dynamic since these twofragments only contain 8 nt for complementation. In this first stepenthalpy plays a key role. Immediately following the first step, asecond step occurs at a very high association rate, resulting in arapid formation of the 3WJ seemingly a one-step reaction with aremarkably low Gibbs free energy (DG�) of �28 kcal/mol, and ahigh melting temperature (TM) of 59.3 �C [55]. In the second step,formation of the 3WJbc complex generated a dimer with a fixedangle and two protruding helices, presenting an increased 17 nt forbinding of 3WJa. The addition of the 3WJa strand results in locking

Fig. 4. RNA nanoparticles with tunable (A) thermal stability, (B) chemical stability and (C) m2014 American Chemical Society; (B) Ref. [53] © 2014 American Chemical Society; (C) Ref.

the unstable 3WJbc complex into a highly stable 3WJ. Consequently,the resulting pRNA-3WJ is more stable than any of the dimer spe-cies as shown in the muchmore rapid association rates and slowestdissociation rate constant [55]. When applying the pRNA-3WJmotif in higher-ordered nanoparticles, diverse thermodynamicproperties were observed. For example, the Tm of RNA squaresincreased corresponding to an increase in size. To adjust thethermo-stability of RNA nanoparticles, the substitution of anucleotide (e.g., DNA, 20-fluorine RNA) in the core strand leads tovariable Tm, as evidenced by the RNA squares with varying corestrand identities creating hybrid base pairings (Fig. 4A) [53]. It hasbeen shown that chemical modifications on the ribose (e.g., 20-F, 20-OMe, locked nucleic acid), or phosphate backbone (e.g., phosphor-othioate linkage) improve the stability of RNA constructs enzy-matically and thermodynamically. Among these modifications, LNAshows the strongest potential in enhancing the thermodynamicstability of RNA nanoparticles as dictated by a significantlyincreased Tm [76].

RNA nanoparticles with tunable chemical stabilities

Themajor difference between DNA and RNA is the 20 location onthe ribose, aneOH for RNA and aneH for DNA. The susceptibility ofnative RNA to nuclease degradation hindered its in vivo applica-tions. To address this issue, chemical modifications to RNA such as20-fluorine (20-F) have shown improved resistance to degradationcaused by cellular nucleases [77]. 20-F pyramidine (C and U)modified RNA drastically enhances chemical and enzymatic sta-bility, rendering a longer half-life of nanoparticles under

echanical stability. Figure adapted and reproduced with permission from: (A) Ref. [53] ©

[82] © AAAS.

Table 1RNA nanoparticles for successful in vivo specific targeting.

Strategy Related marker RNA nanoparticle Model Ref

Aptamer EGFR pRNA-3WJ; RNAtetrahedron

Breast cancer [18,67]

PSMA pRNA-3WJ Prostate cancer [20]Folic acid FR pRNA-X KB xenograft [48]

FR pRNA-3WJ Colorectal cancer; [49]Glioblastoma; [21]Gastric cancer; [19]Head & neck cancer [14]

Oculardelivery

pRNA-X Retina cells [110]pRNA-3WJ Cornea [110]

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

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physiological conditions (Fig. 4B). By changing the ratio of 20-Fmodified RNA present in nanoparticles, different stabilities orsusceptibilities to RNases were achieved, potentially allowing fortemporally-controlled degradation of RNA nanostructures and thuscargo release [53].

RNA nanoparticles with controllable mechanical properties

Unlike mechanically stable proteins [78], nucleic acid structuresgenerally exhibit much lower mechanic stability due in part to lackof long range interactions in their tertiary structure [79e81].Interestingly, pRNA-3WJ showed exceptional mechanical anisot-ropywithstanding forces up to 220 ± 78 pN along the H1eH3 portalaxis upon Mg2þ binding while low mechanical anisotropy wasobserved in the absence of Mg2þ. This reversible modulation ofmechanical stability makes pRNA-3WJ a novel structure for con-struction of biomaterials with controllable mechanical resistance(Fig. 4C) [82].

The elasticity of nanoparticles has recently been viewed as aviable parameter to impact the cellular uptake and intracellulardelivery [83]. Elastic deformation of soft nanoparticles significantlyfacilitates uptake during phagocytosis while a clathrin-mediatedmechanism dominates the internalization of stiff nanoparticles[23,84]. RNA nanoparticles were demonstrated to be of desirableelasticity (unpublished data), rendering them favorable for defor-mation in the process of overcoming a series of biological barriers.

RNA nanoparticles with variable immunomodulation effects

The immune system plays a critical role in defending organismsfrom foreign matter and keeping homeostasis in the human body.Inappropriate immune response or hypoimmunity can lead toadverse side effects. Controlling immune response to RNA nano-particles is critical for developing safe and effective RNA thera-peutics. Recent studies suggest that the immune response elicitedby RNA nanoparticles is highly dependent on RNA sequence,chemical modifications, size, and shape. The pRNA nanoparticlescan be designed to reach two opposite effects: completely non-immunogenic [19,24,52] to serve as vehicles for the delivery oftherapeutics for disease treatment, or strongly immunogenicleading to induction of TNF-a, IL-6 and other cytokines [52], thusserving as potent vaccine adjuvants or reagents for cancer immu-notherapy [85].

Achieving specific targeting of tumors while avoidingentrapment 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 itsrandom nature and lack of control [86]. In addition, due to tumorheterogeneities, not all cancers exhibit EPR effects, thus losing drugefficacy in some tumors [87]. Clinical data remains unclear onwhether the well-documented EPR effect in small animal modelsalso exists in humans [88]. Nonetheless, the major challenge incancer research remains the non-specific accumulation of thera-peutic nanoparticles in healthy vital organs, i.e. liver, lungs, kidneys,and spleen. This low specificity reduces the fraction of nano-particles that reach tumors, while increasing the toxicity and sideeffects [62,89]. Controlled “active” targeting is desirable for suc-cessful treatment strategies to effectively block tumor progressionand prevent metastasis.

Aptamers refer to single-stranded DNA or RNA moleculesselected from an in vitro evolution method [90]. These nucleic acids

typically form specific 3D structures with high-affinity to specificcell surface markers via three-dimensional interactions includinghydrophobic and electrostatic interactions, hydrogen bonding, andvan der Waals forces [91]. With the development of in vitroevolutionary methods (termed SELEX) for the discovery of oligo-nucleotides that bind to protein targets [92], tens of RNA aptamershave been identified for various cell surface receptors (e.g., EGFR[93], PSMA [94], HER2 [95], EpCAM [96], CD4 [97]). After binding tothe receptor, the aptamers can then internalize into the cells viareceptor-mediated endocytosis [98]. However, most aptamersthemselves are susceptible to renal filtration due to a lower mo-lecular mass compared to the cutoff molecular mass of the renalglomerulus [99]. Introducing RNA aptamers to RNA nanoparticlecomplexes increases the circulation time while lowering the off-target effect of RNA nanoparticles. To construct RNA nanoparticlesharboring aptamers, functional sequences are fused into corestrands without affecting their original folding [17]. For example,fusion of an EGFR-targeting aptamer (EGFRapt) with one of the 3WJstrands produces a thermodynamically stable 3WJ-EGFRaptchimera structure with high binding affinity to EGFR over-expressing cancer cells [18]. Similarly, the PSMA aptamer wasincorporated into pRNA-3WJ through bottom-up construction. ThePSMA aptamer-tethered pRNA-3WJ nanoparticles showed specifictargeting to the LNCaP xenograft tumor compared to their coun-terpart nanoparticles without a PSMA aptamer [20].

As an alternative, chemical ligands can be incorporated as tar-geting moieties. As a common tumor-enriched antigen, the folatereceptor (FR) has been identified as a tumor maker, especially inepithelial carcinomas. Folate, the ligand of FR, binds to its receptorwith high affinity [100]. In early drug delivery studies, folate wasconjugated to cytotoxic agents such as chemical drugs and proteintoxins [101]. The folate-tethered macromolecules show enhanceddelivery to folate-expressing cancer cells in vitro in almost all sit-uations tested. However, folate-mediated macromolecular target-ing 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 tonegatively-charged cell membrane [103]. Functionalization offolate or other targeting molecules on RNA nanoparticles signifi-cantly enhances the specific recognition of nanoparticles to targetcells [14,19,21,48,49].

Favorable biodistribution of RNA nanoparticles in vivo for efficacioustreatment

In vitro targeting to cancer cells has been achieved by manynanoparticles; however, site-specific delivery of therapeuticsin vivo faces formidable challenges. Upon intravenous adminis-tration, drug-containing nanoparticles encounter a series of bio-logical barriers. Under physiological conditions, nanoparticles

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Fig. 6. Specific targeting and accumulation of pRNA-X nanoparticles to KB cellsxenograft. (A) Whole-body imaging at different time points. (B) Internal organ imag-ing. Figure adapted and reproduced with permission from Ref. [48] © 2012 Elsevier.

C. Xu et al. / Cancer Letters 414 (2018) 57e70 65

undergo rapid opsonization and subsequent sequestration byresident macrophages of the mononuclear phagocyte system(MPS), resulting in significant accumulation of nanoparticles inhealthy organs such as the liver and spleen [104,105]. To overcomethese obstacles, RNA nanoparticles were exploited to reach spe-cific target sites and bypass the normal organ accumulation andrenal clearance (Table 1). As examples, an RNA aptamer targetingepidermal growth factor receptor (EGFR) was anchored on pRNA-3WJ nanoparticles (Fig. 7A) for specific targeting to triple negativebreast cancer (TNBC) (Fig. 5A). Specific targeting and retention ofRNA nanoparticles in tumors was evidenced by the histologicalassay of tumor frozen cross sections using fluorescence confocalmicroscopy (Fig. 7B). Compared to the control group, treatmentwith pRNA-3WJ-EGFR displayed remarkable accumulation at thetumor site without non-specific accumulation in healthy organs[18]. Similarly, 3D RNA tetrahedron nanoparticles decorated withEGFR aptamers showed tumor-specific enrichment in orthotopicMDA-MB-231 tumor-bearing mice after systemic administration[67]. Another successful aptamer-guided in vivo targeting isexemplified by pRNA-3WJ nanoparticles harboring anti-prostate-specific membrane antigen (PSMA) RNA aptamers (Fig. 5D) [20].An alternative strategy to improve the targeted delivery of nano-medicine into solid tumors is folate conjugation on pRNA-3WJnanoparticles. The pRNA-3WJ-folate conjugates have been suc-cessfully exploited in various FR overexpressed cancer typesincluding 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 thecourse of time, pRNA-X nanoparticles harboring folate and fluo-rophore were systemically injected into athymic nude micebearing KB cell xenografts. At different time points, whole-bodyimaging showed the significant accumulation of RNA nano-particles in tumors within 4 h (Fig. 6A). The organ imaging at 8-htime point indicated the specific localization of pRNA-X nano-particles in tumor without entrapment in the normal organs(Fig. 6B) [48]. Together, several RNA nanoparticle platforms havedemonstrated the superior ability of RNA nanoparticles to lead toselective targeting of diseased tissues over other nanotechnologyplatforms. This targeting overcomes a major hurdle that iscurrently limiting FDA approved therapeutics and will advance thepharmaceutical field in the future.

Despite the favorable distribution of RNA nanoparticles in thetumor xenograft model, accumulation of drug-containing nano-particles in metastases is especially important. As the most lethalaspect of cancer, metastasis of cancer cells is one of the greatestchallenges in cancer treatment today due to their small size, highmultiplicity, 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 exclusivelytargeting metastatic cancers is urgently needed. Rychahou et alreported the specific delivery of folate-conjugated pRNA nano-particles into colorectal cancer metastases (Fig. 5G). After systemicinjection, the RNA nanoparticles bind to CRC liver, lung, and lymphnode metastases without accumulation in normal liver, lung, andother healthy organs [49].

To validate the targeting, the therapy effects of RNA nano-particles harboring therapeutic agents were evaluated in tumor-bearing mice. EGFRapt-guided delivery of anti-miR21 displayed

Fig. 5. Specific targeting of RNA nanoparticles to various xenografts, metastasis, and ocular ticancer; (D) Prostate cancer; (E) Colorectal cancer; (F) Head & neck cancer; (G) CRCmetastasisnanoparticles. (H) Ocular fluorescence imaging of the eye after subconjunctival injection oImpact Journals; (B) Ref. [18] © 2015 American Chemical Society; (C) Ref. [19] © 2015 Macmill© 2015 American Chemical Society; (F) Ref. [14] © 2013 RNA Society; (G) Ref. [49] © 2015 Amreferences to colour in this figure legend, the reader is referred to the web version of this

efficacious inhibition of tumor growth in a TNBCmouse model [19].As shown in the tumor growth curve (Fig. 7C) and luminescencesignal (Fig. 7D), treatment of 5 doses of 3WJ-EGFRapt/anti-miRNA21 resulted in sustained inhibition of tumor growthcompared to control group. The knockdown effect ofmiRNA-21wasalso 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 highly efficient inhibi-tion of prostate cancer [20]. Chemical ligands such as folate haveproved to successfully deliver siRNA to glioblastoma, leading tosignificant gene silencing within the luciferase gene expressingbrain tumors [21].

The favorable in vivo pharmacokinetics of RNA nanoparticlesalso yield potential benefits for ocular disease treatment. Currentocular delivery of therapeutics such as oligonucleotides to theposterior segment of the eye requires repeated intravitreal in-jections, that involves severe side effects such as intraocularbleeding, infection and retinal detachment [108]. Specific deliveryof therapeutics to the posterior segment of the eye is desirable yetchallenging [109]. Subconjunctival injection of pRNA-3WJ, pRNA-Xnanoparticles, and double-stranded RNA (dsRNA) showed differentPK profiles. Cell internalization was observed in the cornea withpRNA-3WJ and pRNA-X nanoparticles. However, only the pRNA-Xnanoparticles could enter the retina (Fig. 5H) [110]. These

ssues. Whole-body and organ imaging of (A) Brain cancer; (B) Breast cancer; (C) Gastricin the lung, liver, lymph node and bones. Green: GFP-cancer cells; blue: DAPI; red: RNAf pRNA-X. Figure adapted and reproduced with permission from: (A) Ref. [21] © 2015an Publishers Limited, part of Springer Nature; (D) Ref. [20] © 2016 Elsevier; (E) Ref. [49]erican Chemical Society; (H) Ref. [110] © 2013 Springer US. (For interpretation of the

article.)

Fig. 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 5injections. (*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-regulationof miRNA21 after treatment, resulting in up-regulation of two target genes PTEN and PDCD4. RQ: relative quantification. Figures adapted with permission from Ref. [18] © 2015American Chemical Society. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C. Xu et al. / Cancer Letters 414 (2018) 57e7066

pharmacokinetic studies provided useful information on the dis-tribution of drugs in the eye and in the development of novel drugdelivery methods.

In general, the favorable biodistribution of RNA nanoparticles isdue to several reasons. The negative charge of RNA minimizesnonspecific binding to negatively charged cell membranes, thusreducing the toxicity associated with normal cells. RNA nano-particles are homogenous with defined structure, size, and stoi-chiometry and thus can avoid nonspecific side effects. RNAnanoparticles are 10e50 nm in size and sufficient to harbor siRNAs,ribozymes, miRNAs, and RNA aptamers [111]. They are large enoughto decrease rapid renal excretion, yet small enough to enter cells byreceptor-mediated endocytosis, while avoiding entrapment by liverKupffer cells and lung/spleen macrophages. The elastic nature andbranched ratchet shape of our RNA nanoparticles facilitate tumorpenetration and increase EPR effects. This is particularly useful forovercoming mechanical barriers, disorganized vascularization, andhighly immunosuppressive tumor microenvironments. A summaryof the advantageous of RNA nanoparticles for in vivo applications islisted 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 ofRNA nanoparticles

Nanoparticle clearance from the reticuloendothelial system(RES) depends not only on size but also on surface modification andcan vary significantly among the different properties of chemicalsconjugated to nanoparticles [61,112]. Tremendous efforts have beenmade to increase water solubility of nanoparticles for longer cir-culation such as polyethylene glycol (PEG) grafting on nano-particles [113,114]. Studies have shown that nanoparticle size andsurface composition (e.g., hydrophobicity) are very important pa-rameters in defining the composition of the formed protein corona.More hydrophobic nanoparticles bound more significant amountsof protein and the association and dissociation rates of boundproteins were clearly dependent on the hydrophobicity of theparticles [115,116]. RNA, as a natural water-soluble polymer, hasbeen engineered as multifunctional nanoparticles by decoratingwith fluorophores, targeting ligands, and therapeutic agents.However, little is known about the effects of conjugating hydro-phobic fluorophores on plasma protein binding or organ accumu-lation. Jasinski et al investigated the changes in vital organaccumulation patterns resulting from surface hydrophobicityvariation. Three fluorophores of different hydrophobicities wereconjugated to the 3WJ RNA nanoparticle to serve as model chem-icals: Cyanine5.5 (Cy5.5); Sulfonated-Cyanine 5.5 (SulfoCy5.5); andAlexaFluor700. RNA nanoparticles with a conjugated fluorophore(3WJ-fluor) exhibited decreased fluorescent signal in vital organs,especially in lung (Lu) and liver (Lv), when compared to that offluorophore alone. This is a strong indication of less accumulationin vital organs for 3WJ-fluor nanoparticles [117].

As shown here, the biological impacts of RNA nanoparticles areclosely related to the modified chemicals following systemicadministration. While changes in RNA nanoparticle protein bindingmight be difficult to predict, understanding the effects of conju-gating hydrophobic drugs on solubility, and in turn in vivo

C. Xu et al. / Cancer Letters 414 (2018) 57e70 67

biodistribution can help researchers develop RNA-based thera-peutics in future clinical settings.

Excretion and clearance of RNA nanoparticles

As growing evidence has demonstrated the favorable bio-distribution of rationally designed RNA nanoparticles in vivo, abasic understanding of how RNA nanoparticles are eliminated isimportant for their future clinical applications. In general, nano-particles undergo excretion and clearance by renal filtration, withexcretion into the urine, or hepatobiliary processing, with excretioninto the bile [118,119]. Based on the urine assay of 3WJ nano-particles, it is suspected that kidney filtration into the urine is themajor excretion pathway for RNA 3WJ nanoparticles [76]. Thoughin vitro serum stability assays and tumor pharmacokinetic modelshave characterized the biological and distribution effects of RNAnanoparticles, more accurate quantitative assays are needed tostreamline in vivo studies.

Conditional endosomal escape of RNA nanoparticles

Specific targeting and favorable in vivo biodistribution of RNAnanoparticles ensure the delivery of therapeutic agents to thetarget cancer cells; however, their intracellular bioavailability isalso critical for efficacious therapy. Nanoparticles are typicallytaken up into cells via endocytic pathwaywhere they are entrappedin endosomes and are then subsequently trafficked into lysosomalcompartments. The acidic environment of specific enzymes in thelysosome leads to significant degradation of the therapeutics [120].Recent studies on nanoparticle intracellular trafficking indicate thatthe siRNA delivery efficiency is significantly hindered by insuffi-cient endosomal escape [121]. Hence, a rate-limiting yet chal-lenging barrier in achieving an effective therapy is to facilitate theendosomal escape and ensure cytosolic delivery of thetherapeutics.

Despite the success of the RNA nanoparticle in cancer therapy,little is known about its internalization pathway and subsequentintracellular trafficking. Ample in vivo cancer treatment outcomesindirectly suggested successful endosomal escape of RNA nano-particles is required to exert therapeutic effects. For example, pRNA-3WJ has been exploited for efficient delivery of anti-miRNA withmore significant gene regulation effects compared to that inducedby siRNA [18].

Different strategies have been developed to facilitate release ofnanoparticles from endosomes before lysosomal degradation [122].General mechanisms are proposed such as pore formation in theendosomal membrane, “proton sponge” effect of protonable groupsand fusion into the lipid bilayer of the endosome [123,124]. Of note,the protonable molecules induce an extensive inflow of ions andwater into the endosome, leading to osmotic swelling and subse-quent rupture of the endosomal membrane. Imidazole and otherpH sensitive groups (e.g., hydrazine, maleic amides, amino esters)are typically incorporated into the nanoparticles for an enhancedpH buffering capacity [125e127]. As many conjugation methods forend-labeling of RNA have been established, conjugation of endo-some disrupting compounds to RNA nanoparticles poses remark-able impacts in surmounting endosomal barriers.

Challenges and limitations of RNA nanotechnology forbiomedical applications

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

One of the major bottlenecks for RNA nanotechnology in ther-apeutic applications is the large-scale production and high cost ofnanoparticle construction. RNA oligonucleotides prepared by solidphase synthesis are in maximum 80 nt long, preventing nano-particle designers from using longer sequences in the modulardesign of RNA nanostructure. The high cost of RNA synthesis alsolimits the industrial scale production of RNA nanoparticles thoughefforts in improving chemical synthesis have been made [128,129].To improve the recovery of RNA nanoparticles, purificationmethods such as preparative ultracentrifugation have been devel-oped to overcome the low yield of gel electrophoresis and HPLCpurification [54]. Referring to the progressively reduced cost of DNAoligosynthesis over the years [130], it is expected that the cost ofRNA production will gradually decrease with the improvements inchemical synthesis efficiency and production facilities developed inindustries [128,129]. Production of RNA by biological approachessuch as bacterial fermentation in combination with intracellularassembly of RNA nanoparticles has also been proposed [10].

The active targeting strategy deployed on RNA nanotechnologyis successful yet challenging. Given the heterogeneous nature ofcancer, a specific targeting ligand is required for each cancer type[131,132]. However, the number of available RNA aptamers and li-gands is limited and insufficient. Rapid screening methods derivedfrom SELEX have shown considerable promise to keep up with theemergence of RNA-based therapeutics [111]. Computationalmethods such as structural modeling and prediction have shown tofacilitate the experimental screening process of aptamer [133e135].

With the advancement in material science and engineering, thenanoparticle system offers a promising opportunity for targetedcancer therapy. However, a recent report showed that only 0.7%(median) of the administered nanoparticle dose accumulates intumor based on the analysis of publications in “nanoparticle de-livery” over last 10 years [136]. This finding brought up concerns forall nanoparticle-based specific targeting and drug delivery systems.Current findings on RNA nanoparticle in vivo behaviors such as lowentrapment in healthy vital organs, rich accumulation in tumorsand high efficiency in inhibiting cancer growth have provided ev-idence that a significant amount of administered RNA nanoparticlesaccumulate in tumors. However, as RNA nanotechnology for cancertreatment is merely at a starting point, multi-parametric studiesand quantification analysis are needed for better understandingand interpretation of the in vivo behavior of RNA nanoparticles withdifferent synthetic and biological identities. With more funda-mental studies on the pharmacokinetics of RNA nanoparticles,optimized design on their physicochemical properties includingsize, shape, and chemical modification will be guided for improvedspecific targeting and delivery efficiency.

Conclusions

Nanoparticles have evolved considerably in preclinical andclinical studies for cancer targeting and therapy in the past twodecades; it is critical to overcome the biological barriers that hinderthe in vivo targeting and intracellular delivery of nanoparticles.Though present-day nanoparticle-based therapy is extendingbeyond the confines of convention and transitioning toward morerational design, there is the growing realization that the distinctobstacles are by no means insurmountable. RNA nanotechnology-based therapy shed new light on innovative design of therapeu-tics for clinic translation.

As highlighted here, two major active targeting strategiesimplemented on pRNA-3WJ based RNA nanoparticles to specificcancer were demonstrated in different tumor models. Given thehighly heterogeneous and continuously evolving nature of the tu-mor, the development of tumor-specific RNA nanoparticles benefits

C. Xu et al. / Cancer Letters 414 (2018) 57e7068

the optimized delivery in personalized medicine. In virtue of thenature of RNA as a building block in nanoparticle design, physico-chemical properties of RNA complexes are also tunable. Under-standing the effects of RNA nanoparticle size, shape, and surfacechemistry on its biodistribution in biological systems will assist inthe rational design of entities that are capable of sequential nego-tiation of biological obstacles for efficacious, site-specific delivery.Increasing evidence has revealed the correlation of physicochem-ical properties of RNA nanoparticles with their in vivo PK profiles.However, an in-depth understanding of their intracellular traf-ficking as well as endocytic pathway are needed. Ultimately, thismay not only lead to the successful translation of novel RNA-basedtherapeutics, but will also guide the design of precision nano-medicine towards treatment of diseases.

Acknowledgements

The research in P.G.’s lab was supported by the National In-stitutes of Health [R01EB019036, U01CA151648, U01CA207946] toPeixuan Guo as well as [P50CA168505, R21CA209045] and DODAward [W81XWH-15-1-0052] to Dan Shu.

Conflict of interest

P.G.'s Sylvan G. Frank Endowed Chair position in Pharmaceuticsand Drug Delivery is funded by the CM Chen Foundation. P.G. is theconsultant of Oxford Nanopore Technologies and Nanobio DeliveryPharmaceutical Co. Ltd, as well as the cofounder of Shenzhen P&ZBio-medical Co. Ltd and its subsidiary US P&Z Biological TechnologyLLC. F.H. is employed by Nanobio Delivery Pharmaceutical, Co. Ltd.,which specializes in RNA nanoparticle production.

References

[1] E. Blanco, H. Shen, M. Ferrari, Principles of nanoparticle design for over-coming biological barriers to drug delivery, Nat. Biotechnol. 33 (2015)941e951.

[2] V.P. Chauhan, R.K. Jain, Strategies for advancing cancer nanomedicine, Nat.Mater. 12 (2013) 958e962.

[3] R.K. Jain, T. Stylianopoulos, Delivering nanomedicine to solid tumors, Nat.Rev. Clin. Oncol. 7 (2010) 653e664.

[4] S. Mitragotri, P.A. Burke, R. Langer, Overcoming the challenges in adminis-tering biopharmaceuticals: formulation and delivery strategies, Nat. Rev.Drug Discov. 13 (2014) 655e672.

[5] J. Shi, P.W. Kantoff, R. Wooster, O.C. Farokhzad, Cancer nanomedicine:progress, challenges and opportunities, Nat. Rev. Cancer 17 (2017) 20e37.

[6] C.M. Dawidczyk, C. Kim, J.H. Park, L.M. Russell, K.H. Lee, M.G. Pomper, et al.,State-of-the-art in design rules for drug delivery platforms: lessons learnedfrom FDA-approved nanomedicines, J. Control Release 187 (2014) 133e144.

[7] J. Shi, Z. Xiao, N. Kamaly, O.C. Farokhzad, Self-assembled targeted nano-particles: evolution of technologies and bench to bedside translation, Acc.Chem. Res. 44 (2011) 1123e1134.

[8] P. Guo, The emerging field of RNA nanotechnology, Nat. Nanotechnol. 5(2010) 833e842.

[9] D. Shu, Y. Shu, F. Haque, S. Abdelmawla, P. Guo, Thermodynamically stableRNA three-way junctions for constructing multifuntional nanoparticles fordelivery of therapeutics, Nat. Nanotechnol. 6 (2011) 658e667.

[10] D. Jasinski, F. Haque, D.W. Binzel, P. Guo, Advancement of the emerging fieldof RNA nanotechnology, ACS Nano 11 (2017) 1142e1164.

[11] Y. Shu, F. Pi, A. Sharma, M. Rajabi, F. Haque, D. Shu, et al., Stable RNAnanoparticles as potential new generation drugs for cancer therapy, Adv.Drug Deliv. Rev. 66C (2014) 74e89.

[12] H. Li, T. Lee, T. Dziubla, F. Pi, S. Guo, J. Xu, et al., RNA as a stable polymer tobuild controllable and defined nanostructures for material and biomedicalapplications, Nano Today 10 (2015) 631e655.

[13] S. Lemieux, F. Major, RNA canonical and non-canonical base pairing types: arecognition method and complete repertoire, Nucleic Acids Res. 30 (2002)4250e4263.

[14] Y. Shu, F. Haque, D. Shu, W. Li, Z. Zhu, M. Kotb, et al., Fabrication of 14different RNA nanoparticles for specific tumor targeting without accumula-tion in normal organs, RNA 19 (2013) 766e777.

[15] H. Zhang, P. Guo, Single molecule photobleaching (SMPB) technology forcounting of RNA, DNA, protein and other molecules in nanoparticles andbiological complexes by TIRF instrumentation, Methods 67 (2014) 169e176.

[16] R. Reif, F. Haque, P. Guo, Fluorogenic RNA nanoparticles for monitoring RNAfolding and degradation in real time in living cells, Nucleic Acid. Ther. 22 (6)(2013) 428e437.

[17] D. Shu, E. Khisamutdinov, L. Zhang, P. Guo, Programmable folding of fusionRNA complex driven by the 3WJ motif of phi29 motor pRNA, Nucleic AcidsRes. 42 (2013) e10.

[18] D. Shu, H. Li, Y. Shu, G. Xiong, W.E. Carson, F. Haque, et al., Systemic deliveryof anti-miRNA for suppression of triple negative breast cancer utilizing RNAnanotechnology, ACS Nano 9 (2015) 9731e9740.

[19] D. Cui, C. Zhang, B. Liu, Y. Shu, T. Du, D. Shu, et al., Regression of gastric cancerby systemic injection of RNA nanoparticles carrying both ligand and siRNA,Sci. Rep. 5 (2015) 10726.

[20] D. Binzel, Y. Shu, H. Li, M. Sun, Q. Zhang, D. Shu, et al., Specific delivery ofMiRNA for high efficient inhibition of prostate cancer by RNA nanotech-nology, Mol. Ther. 24 (2016) 1267e1277.

[21] T.J. Lee, F. Haque, D. Shu, J.Y. Yoo, H. Li, R.A. Yokel, et al., RNA nanoparticles asa vector for targeted siRNA delivery into glioblastoma mouse model, Onco-target 6 (2015) 14766e14776.

[22] F. Pi, H. Zhang, H. Li, V. Thiviyanathan, D.G. Gorenstein, A.K. Sood, et al., RNAnanoparticles harboring annexin A2 aptamer can target ovarian cancer fortumor-specific doxorubicin delivery, Nanomedicine 13 (2016) 1183e1193.

[23] A.C. Anselmo, S. Mitragotri, Impact of particle elasticity on particle-baseddrug delivery systems, Adv. Drug Deliv. Rev. 108 (2017) 51e67.

[24] S. Abdelmawla, S. Guo, L. Zhang, S. Pulukuri, P. Patankar, P. Conley, et al.,Pharmacological characterization of chemically synthesized monomericpRNA nanoparticles for systemic delivery, Mol. Ther. 19 (2011) 1312e1322.

[25] W.W. Grabow, L. Jaeger, RNA self-assembly and RNA nanotechnology, Ac-counts Chem. Res. 47 (2014) 1871e1880.

[26] D. Shu, W.D. Moll, Z. Deng, C. Mao, P. Guo, Bottom-up assembly of RNA arraysand superstructures as potential parts in nanotechnology, Nano Lett. 4(2004) 1717e1723.

[27] W.W. Grabow, P. Zakrevsky, K.A. Afonin, A. Chworos, B.A. Shapiro, L. Jaeger,Self-Assembling RNA nanorings based on RNAI/II inverse kissing complexes,Nano Lett. 11 (2011) 878e887.

[28] N.B. Leontis, A. Lescoute, E. Westhof, The building blocks and motifs of RNAarchitecture, Curr. Opin. Struct. Biol. 16 (2006) 279e287.

[29] N.B. Leontis, E. Westhof, Analysis of RNA motifs, Curr. Opin. Struct. Biol. 13(2003) 300e308.

[30] L. Jaeger, E. Westhof, N.B. Leontis, TectoRNA: modular assembly units for theconstruction of RNA nano-objects, Nucleic Acids Res. 29 (2001) 455e463.

[31] A. Chworos, I. Severcan, A.Y. Koyfman, P. Weinkam, E. Oroudjev,H.G. Hansma, et al., Building programmable jigsaw puzzles with RNA, Sci-ence 306 (2004) 2068e2072.

[32] I. Severcan, C. Geary, E. V, A. C, L. Jaeger, Square-shaped RNA particles fromdifferent RNA folds, Nano Lett. 9 (2009) 1270e1277.

[33] L. Jaeger, N.B. Leontis, Tecto-RNA: one dimensional self-assembly throughtertiary interactions, Angew. Chem. Int. Ed. Engl. 39 (2000) 2521e2524.

[34] J. Ishikawa, H. Furuta, Y. Ikawa, RNA Tectonics (tectoRNA) for RNA nano-structure design and its application in synthetic biology, Wiley. Interdiscip.Rev. RNA 4 (2013) 651e664.

[35] I. Severcan, C. Geary, A. Chworos, N. Voss, E. Jacovetty, L. Jaeger,A polyhedron made of tRNAs, Nat. Chem. 2 (2010) 772e779.

[36] M. Abraham, O. Dror, R. Nussinov, H.J. Wolfson, Analysis and classification ofRNA tertiary structures, RNA 14 (2008) 2274e2289.

[37] E. Bindewald, R. Hayes, Y.G. Yingling, W. Kasprzak, B.A. Shapiro, RNA-Junction: a database of RNA junctions and kissing loops for three-dimensional structural analysis and nanodesign, Nucleic Acids Res. 36(2008) D392eD397.

[38] E. Bindewald, C. Grunewald, B. Boyle, M. O'Connor, B.A. Shapiro, Computa-tional strategies for the automated design of RNA nanoscale structures frombuilding blocks using NanoTiler, J. Mol. Graph. Model. 27 (2008) 299e308.

[39] F. Jossinet, T.E. Ludwig, E. Westhof, Assemble: an interactive graphical tool toanalyze and build RNA architectures at the 2D and 3D levels, Bioinformatics26 (2010) 2057e2059.

[40] H.M. Martinez, J.V. Maizel, B.A. Shapiro, RNA2D3D: a program for generating,viewing, and comparing 3-dimensional models of RNA, J. Biomol. Struct. Dyn.25 (2008) 669e683.

[41] A. Busch, R. Backofen, INFO-RNAea server for fast inverse RNA foldingsatisfying sequence constraints, Nucleic Acids Res. 35 (2007) W310eW313.

[42] J.N. Zadeh, C.D. Steenberg, J.S. Bois, B.R. Wolfe, M.B. Pierce, A.R. Khan, et al.,NUPACK: analysis and design of nucleic acid systems, J. Comput. Chem. 32(2011) 170e173.

[43] C. Geary, P.W. Rothemund, E.S. Andersen, A single-stranded architecture forcotranscriptional folding of RNA nanostructures, Science 345 (2014)799e804.

[44] K.A. Afonin, R. Desai, M. Viard, M.L. Kireeva, E. Bindewald, C.L. Case, et al., Co-transcriptional production of RNA-DNA hybrids for simultaneous release ofmultiple split functionalities, Nucleic Acids Res. 42 (2014) 2085e2097.

[45] K.A. Afonin, M. Kireeva, W.W. Grabow, M. Kashlev, L. Jaeger, B.A. Shapiro, Co-transcriptional assembly of chemically modified RNA nanoparticles func-tionalized with siRNAs, Nano. Lett. 12 (2012) 5192e5195.

[46] J.B. Lee, J. Hong, D.K. Bonner, Z. Poon, P.T. Hammond, Self-assembled RNAinterference microsponges for efficient siRNA delivery, Nat. Mater 11 (2012)316e322.

C. Xu et al. / Cancer Letters 414 (2018) 57e70 69

[47] D. Han, Y. Park, H. Kim, J.B. Lee, Self-assembly of free-standing RNA mem-branes, Nat. Commun. 5 (2014) 4367.

[48] F. Haque, D. Shu, Y. Shu, L. Shlyakhtenko, P. Rychahou, M. Evers, et al.,Ultrastable synergistic tetravalent RNA nanoparticles for targeting to can-cers, Nano Today 7 (2012) 245e257.

[49] P. Rychahou, F. Haque, Y. Shu, Y. Zaytseva, H.L. Weiss, E.Y. Lee, et al., Deliveryof RNA nanoparticles into colorectal cancer metastases following systemicadministration, ACS Nano 9 (2015) 1108e1116.

[50] Y. Shu, D. Shu, F. Haque, P. Guo, Fabrication of pRNA nanoparticles to delivertherapeutic RNAs and bioactive compounds into tumor cells, Nat. Protoc. 8(2013) 1635e1659.

[51] E.F. Khisamutdinov, D.L. Jasinski, P. Guo, RNA as a boiling-resistant anionicpolymer material to build robust structures with defined shape and stoi-chiometry, ACS Nano 8 (2014) 4771e4781.

[52] E. Khisamutdinov, H. Li, D. Jasinski, J. Chen, J. Fu, P. Guo, Enhancing immu-nomodulation on innate immunity by shape transition among RNA triangle,square, and pentagon nanovehicles, Nucleic Acids Res. 42 (2014)9996e10004.

[53] D. Jasinski, E.F. Khisamutdinov, Y.L. Lyubchenko, P. Guo, Physicochemicallytunable poly-functionalized RNA square architecture with fluorogenic andribozymatic properties, ACS Nano 8 (2014) 7620e7629.

[54] D. Jasinski, C. Schwartz, F. Haque, P. Guo, Large scale purification of RNAnanoparticles by preparative ultracentrifugation, Methods Mol. Biol. 1297(2015) 67e82.

[55] D.W. Binzel, E.F. Khisamutdinov, P. Guo, Entropy-driven one-step formationof Phi29 pRNA 3WJ from three RNA fragments, Biochemistry 53 (2014)2221e2231.

[56] D.W. Binzel, E. Khisamutdinov, M. Vieweger, J. Ortega, J. Li, P. Guo, Mecha-nism of three-component collision to produce ultrastable pRNA three-wayjunction of Phi29 DNA-packaging motor by kinetic assessment, RNA 22(2016) 1710e1718.

[57] J. Liu, S. Guo, M. Cinier, L.S. Shlyakhtenko, Y. Shu, C. Chen, et al., Fabrication ofstable and RNase-resistant RNA nanoparticles active in gearing the nano-motors for viral DNA packaging, ACS Nano 5 (2011) 237e246.

[58] H.G. Hansma, E. Oroudjev, S. Baudrey, L. Jaeger, TectoRNA and 'kissing-loop'RNA: atomic force microscopy of self-assembling RNA structures, J. Microsc.212 (2003) 273e279.

[59] M. Zuker, Mfold web server for nucleic acid folding and hybridization pre-diction, Nucleic Acids Res. 31 (2003) 3406e3415.

[60] P. Guo, C. Zhang, C. Chen, M. Trottier, K. Garver, Inter-RNA interaction ofphage phi29 pRNA to form a hexameric complex for viral DNA trans-portation, Mol. Cell. 2 (1998) 149e155.

[61] M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K.A. Dawson, Nano-particle size and surface properties determine the protein corona withpossible implications for biological impacts, Proc. Natl. Acad. Sci. U. S. A 105(2008) 14265e14270.

[62] R.C. von, W. Jiang, C.K. Chan, I.L. Weissman, B.Y. Kim, Breaking down thebarriers to precision cancer nanomedicine, Trends Biotechnol. 35 (2017)159e171.

[63] X. Huang, L. Li, T. Liu, N. Hao, H. Liu, D. Chen, et al., The shape effect ofmesoporous silica nanoparticles on biodistribution, clearance, and biocom-patibility in vivo, ACS Nano 5 (2011) 5390e5399.

[64] Z. Ma, J. Bai, Y. Wang, X. Jiang, Impact of shape and pore size of mesoporoussilica nanoparticles on serum protein adsorption and RBCs hemolysis, ACSAppl. Mat. Interfac. 6 (2014) 2431e2438.

[65] K. Muller, D.A. Fedosov, G. Gompper, Margination of micro- and nano-particles in blood flow and its effect on drug delivery, Sci. Rep. 4 (2014) 4871.

[66] Y. Zhao, Y. Wang, F. Ran, Y. Cui, C. Liu, Q. Zhao, et al., A comparison betweensphere and rod nanoparticles regarding their in vivo biological behavior andpharmacokinetics, Sci. Rep. 7 (2017) 4131.

[67] H. Li, K. Zhang, F. Pi, S. Guo, L. Shlyakhtenko, W. Chiu, et al., Controllable self-assembly of RNA tetrahedrons with precise shape and size for cancer tar-geting, Adv. Mater 28 (2016) 7501e7507.

[68] E.F. Khisamutdinov, D.L. Jasinski, H. Li, K. Zhang, W. Chiu, P. Guo, Fabricationof RNA 3D nanoprism for loading and protection of small RNAs and modeldrugs, Adv. Mater. 28 (2016) 100079e100087.

[69] A. Albanese, P.S. Tang, W.C. Chan, The effect of nanoparticle size, shape, andsurface chemistry on biological systems, Annu. Rev. Biomed. Eng. 14 (2012)1e16.

[70] H. Zhang, J.A. Endrizzi, Y. Shu, F. Haque, C. Sauter, L.S. Shlyakhtenko, et al.,Crystal structure of 3WJ core revealing divalent ion-promoted thermosta-bility and assembly of the Phi29 hexameric motor pRNA, RNA 19 (2013)1226e1237.

[71] A. Sharma, F. Haque, F. Pi, L. Shlyakhtenko, B.M. Evers, P. Guo, Controllableself-assembly of RNA dendrimers, Nanomed. Nanotechnol. Biol. Med. 12(2015) 835e844.

[72] N. Sugimoto, S. Nakano, M. Katoh, A. Matsumura, H. Nakamuta, T. Ohmichi,et al., Thermodynamic parameters to predict stability of RNA/DNA hybridduplexes, Biochemistry 34 (1995) 11211e11216.

[73] M.S. Searle, D.H. Williams, On the stability of nucleic acid structures in so-lution: enthalpy-entropy compensations, internal rotations and reversibility,Nucleic Acids Res. 21 (1993) 2051e2056.

[74] E.A. Lesnik, S.M. Freier, Relative thermodynamic stability of DNA, RNA, andDNA-RNA hybrid duplexes - relationship with base composition and struc-ture, Biochemistry 34 (1995) 10807e10815.

[75] K. Tiemann, J.J. Rossi, RNAi-based therapeutics-current status, challenges andprospects, EMBO Mol. Med. 1 (2009) 142e151.

[76] X. Piao, H. Wang, D. Binzel, P. Guo, Assessment of Thermal Stability ofPhosphorothioate DNA, DNA, RNA, 2'-F RNA and LNA in the Context of Phi29pRNA 3WJ, RNA (2017), https://doi.org/10.1261/rna.063057.117.

[77] C.R. Allerson, N. Sioufi, R. Jarres, T.P. Prakash, N. Naik, A. Berdeja, et al., Fully2'-modified oligonucleotide duplexes with improved in vitro potency andstability compared to unmodified small interfering RNA, J. Med. Chem. 48(2005) 901e904.

[78] C.L. Brooks III, M. Gruebele, J.N. Onuchic, P.G. Wolynes, Chemical physics ofprotein folding, Proc. Natl. Acad. Sci. U. S. A 95 (1998) 11037e11038.

[79] J. Liphardt, B. Onoa, S.B. Smith, I. Tinoco Jr., C. Bustamante, Reversibleunfolding of single RNA molecules by mechanical force, Science 292 (2001)733e737.

[80] W.J. Greenleaf, K.L. Frieda, D.A. Foster, M.T. Woodside, S.M. Block, Directobservation of hierarchical folding in single riboswitch aptamers, Science319 (2008) 630e633.

[81] M. Rief, H. Clausen-Schaumann, H.E. Gaub, Sequence-dependent mechanicsof single DNA molecules, Nat. Struct. Biol. 6 (1999) 346e349.

[82] Z. Xu, Y. Sun, J.K. Weber, Y. Cao, W. Wang, D. Jasinski, et al., Directionalmechanical stability of Bacteriophage phi29 motor's 3WJ-pRNA: extraordi-nary robustness along portal axis, Sci. Adv. 3 (2017) e1601684.

[83] A.C. Anselmo, M. Zhang, S. Kumar, D.R. Vogus, S. Menegatti, M.E. Helgeson, etal., Elasticity of nanoparticles influences their blood circulation, phagocy-tosis, endocytosis, and targeting, ACS Nano 9 (2015) 3169e3177.

[84] X. Banquy, F. Suarez, A. Argaw, J.M. Rabanel, P. Grutter, J.F. Bouchard, et al.,Effect of mechanical properties of hydrogel nanoparticles on macrophagecell uptake, Soft Matter 5 (2009) 3984e3991.

[85] S. Guo, H. Li, M. Ma, J. Fu, Y. Dong, P. Guo, Size, Shape and Sequence-dependent Immunogenicity of RNA Nanoparticles, Mol. Ther. Nucleic Acids(2017), https://doi.org/10.1016/j.omtn.2017.10.010.

[86] V. Torchilin, Tumor delivery of macromolecular drugs based on the EPR ef-fect, Adv. Drug Deliv. Rev. 63 (2011) 131e135.

[87] U. Prabhakar, H. Maeda, R.K. Jain, E.M. Sevick-Muraca, W. Zamboni,O.C. Farokhzad, et al., Challenges and key considerations of the enhancedpermeability and retention effect for nanomedicine drug delivery inoncology, Cancer Res. 73 (2013) 2412e2417.

[88] F. Danhier, To exploit the tumor microenvironment: since the EPR effect failsin the clinic, what is the future of nanomedicine? J. Control Release 244(2016) 108e121.

[89] T.J. Anchordoquy, Y. Barenholz, D. Boraschi, M. Chorny, P. Decuzzi,M.A. Dobrovolskaia, et al., Mechanisms and barriers in cancer nanomedicine:addressing challenges, looking for solutions, ACS Nano 11 (2017) 12e18.

[90] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that bindspecific ligands, Nature 346 (1990) 818e822.

[91] T. Hermann, D.J. Patel, Adaptive recognition by nucleic acid aptamers, Sci-ence 287 (2000) 820e825.

[92] X. Fang, W. Tan, Aptamers generated from cell-SELEX for molecular medi-cine: a chemical biology approach, Acc. Chem. Res. 43 (2010) 48e57.

[93] C.L. Esposito, D. Passaro, I. Longobardo, G. Condorelli, P. Marotta, A. Affuso, etal., A neutralizing RNA aptamer against EGFR causes selective apoptotic celldeath, PLoS ONE 6 (9) (2011) e24071, https://doi.org/10.1371/journal.pone.0024071.

[94] S.E. Lupold, B.J. Hicke, Y. Lin, D.S. Coffey, Identification and characterizationof nuclease-stabilized RNA molecules that bind human prostate cancer cellsvia the prostate-specific membrane antigen, Cancer Res. 62 (2002)4029e4033.

[95] M.Y. Kim, S. Jeong, In vitro selection of RNA aptamer and specific targeting ofErbB2 in breast cancer cells, Nucleic Acid. Ther. 21 (2011) 173e178.

[96] S. Shigdar, J. Lin, Y. Yu, M. Pastuovic, M. Wei, W. Duan, RNA aptamer against acancer stem cell marker epithelial cell adhesion molecule, Cancer Sci. 102(2011) 991e998.

[97] K.A. Davis, Y. Lin, B. Abrams, S.D. Jayasena, Staining of cell surface humanCD4 with 2'-F-pyrimidine-containing RNA aptamers for flow cytometry,Nucleic Acids Res. 26 (1998) 3915e3924.

[98] J. Zhou, J.J. Rossi, Cell-specific aptamer-mediated targeted drug delivery,Oligonucleotides 21 (2011) 1e10.

[99] J. Zhou, J. Rossi, Aptamers as targeted therapeutics: current potential andchallenges, Nat. Rev. Drug Discov. 16 (2017) 181e202.

[100] J. Sudimack, R.J. Lee, Targeted drug delivery via the folate receptor, Adv. DrugDeliv. Rev. 41 (2000) 147e162.

[101] S. Wang, P.S. Low, Folate-mediated targeting of antineoplastic drugs, imag-ing agents, and nucleic acids to cancer cells, J. Control Release 53 (1998)39e48.

[102] Y. Lu, P.S. Low, Folate-mediated delivery of macromolecular anticancertherapeutic agents, Adv. Drug Deliv. Rev. 64 (2012) 342e352.

[103] P. Guo, O. Coban, N.M. Snead, J. Trebley, S. Hoeprich, S. Guo, et al., Engi-neering RNA for targeted siRNA delivery and medical application, Adv. DrugDeliv. Rev. 62 (2010) 650e666.

[104] M. Hadjidemetriou, K. Kostarelos, Nanomedicine: evolution of the nano-particle corona, Nat. Nanotechnol. 12 (2017) 288e290.

[105] M.P. Monopoli, C. Aberg, A. Salvati, K.A. Dawson, Biomolecular coronasprovide the biological identity of nanosized materials, Nat. Nanotechnol. 7(2012) 779e786.

C. Xu et al. / Cancer Letters 414 (2018) 57e7070

[106] G.P. Gupta, J. Massague, Cancer metastasis: building a framework, Cell 127(2006) 679e695.

[107] A. Schroeder, D.A. Heller, M.M. Winslow, J.E. Dahlman, G.W. Pratt, R. Langer,et al., Treating metastatic cancer with nanotechnology, Nat. Rev. Cancer 12(2012) 39e50.

[108] S.S. Lee, M.R. Robinson, Novel drug delivery systems for retinal diseases. Areview, Ophthalmic Res. 41 (2009) 124e135.

[109] A. Urtti, Challenges and obstacles of ocular pharmacokinetics and drug de-livery, Adv. Drug Deliv. Rev. 58 (2006) 1131e1135.

[110] L. Feng, S.K. Li, H. Liu, C.Y. Liu, K. LaSance, F. Haque, et al., Ocular delivery ofpRNA nanoparticles: distribution and clearance after subconjunctival injec-tion, Pharm. Res. 31 (2014) 1046e1058.

[111] P. Guo, F. Haque, B. Hallahan, R. Reif, H. Li, Uniqueness, advantages, chal-lenges, solutions, and perspectives in therapeutics applying RNA nanotech-nology, Nucleic Acid. Ther. 22 (2012) 226e245.

[112] Y. Qie, H. Yuan, C.A. Von Roemeling, Y. Chen, X. Liu, K.D. Shih, et al., Surfacemodification of nanoparticles enables selective evasion of phagocytic clear-ance by distinct macrophage phenotypes, Sci. Rep. 6 (2016) 26269.

[113] Q. He, Z. Zhang, F. Gao, Y. Li, J. Shi, In vivo biodistribution and urinaryexcretion of mesoporous silica nanoparticles: effects of particle size andPEGylation, Small 7 (2011) 271e280.

[114] J.V. Jokerst, T. Lobovkina, R.N. Zare, S.S. Gambhir, Nanoparticle PEGylation forimaging and therapy, Nanomedicine (Lond) 6 (2011) 715e728.

[115] M. Mahmoudi, I. Lynch, M.R. Ejtehadi, M.P. Monopoli, F.B. Bombelli,S. Laurent, Protein�nanoparticle interactions: opportunities and challenges,Chem. Rev. 111 (2011) 5610e5637.

[116] R. Podila, R. Chen, P.C. Ke, J.M. Brown, A.M. Rao, Effects of surface functionalgroups on the formation of nanoparticle-protein corona, Appl. Phys. Lett. 101(2012) 263701.

[117] D.L. Jasinski, H. Yin, Z. Li, P. Guo, The hydrophobic effect from conjugatedchemicals or drugs on in vivo biodistribution of RNA nanoparticles, Hum.Gene Ther. (2017), https://doi.org/10.1089/hum.2017.054.

[118] Y. Yoshioka, K. Higashisaka, S.i. Tsunoda, Y. Tsutsumi, The absorption, dis-tribution, metabolism, and excretion profile of nanoparticles, in: M. Akashi,T. Akagi, M. Matsusaki (Eds.), Engineered Cell Manipulation for BiomedicalApplication, Springer, Japan, Tokyo, 2014, pp. 259e271, 2014.

[119] Y.N. Zhang, W. Poon, A.J. Tavares, I.D. McGilvray, W.C. Chan, Nanoparticle-liver interactions: cellular uptake and hepatobiliary elimination, J. ControlRelease 240 (2016) 332e348.

[120] T.F. Martens, K. Remaut, J. Demeester, S.C. De Smedt, K. Braeckmans, Intra-cellular delivery of nanomaterials: how to catch endosomal escape in the act,Nano Today 9 (2014) 344e364.

[121] M. Dominska, D.M. Dykxhoorn, Breaking down the barriers: siRNA deliveryand endosome escape, J. Cell Sci. 123 (2010) 1183e1189.

[122] D. Ma, Enhancing endosomal escape for nanoparticle mediated siRNA de-livery, Nanoscale 6 (2014) 6415e6425.

[123] L.Y. Chou, K. Ming, W.C. Chan, Strategies for the intracellular delivery ofnanoparticles, Chem. Soc. Rev. 40 (2011) 233e245.

[124] A.K. Varkouhi, M. Scholte, G. Storm, H.J. Haisma, Endosomal escape pathwaysfor delivery of biologicals, J. Control Release 151 (2011) 220e228.

[125] S. Duan, W. Yuan, F. Wu, T. Jin, Polyspermine Imidazole�4, 5�imine, achemically dynamic and biologically responsive carrier system for intracel-lular delivery of siRNA, Angew. Chem. Int. Ed. 51 (2012) 7938e7941.

[126] D.W. Pack, D. Putnam, R. Langer, Design of imidazole-containing endo-somolytic biopolymers for gene delivery, Biotechnol. Bioeng. 67 (2000)217e223.

[127] H.K. Shete, R.H. Prabhu, V.B. Patravale, Endosomal escape: a bottleneck inintracellular delivery, J. Nanosci. Nanotechnol. 14 (2014) 460e474.

[128] T. Lavergne, J.R. Bertrand, J.J. Vasseur, F. Debart, A base-labile group for 2'-OHprotection of ribonucleosides: a major challenge for RNA synthesis, Chem-istry 14 (2008) 9135e9138.

[129] W.S. Marshall, R.J. Kaiser, Recentadvances in the high-speed solid phasesynthesis of RNA, Curr. Opin. Chem. Biol. (2004), https://doi.org/10.1016/j.cbpa.2004.04.012.

[130] S. Kosuri, G.M. Church, Large-scale de novo DNA synthesis: technologies andapplications, Nat. Methods 11 (2014) 499e507.

[131] R.A. Burrell, N. McGranahan, J. Bartek, C. Swanton, The causes and conse-quences of genetic heterogeneity in cancer evolution, Nature 501 (2013)338e345.

[132] R. Fisher, L. Pusztai, C. Swanton, Cancer heterogeneity: implications fortargeted therapeutics, Br. J. Cancer 108 (2013) 479e485.

[133] X. Luo, M. McKeague, S. Pitre, M. Dumontier, J. Green, A. Golshani, Compu-tational approaches toward the design of pools for the in vitro selection ofcomplex aptamers, RNA 16 (2010) 2252e2262.

[134] W.M. Rockey, F.J. Hernandez, S.Y. Huang, S. Cao, C.A. Howell, G.S. Thomas, etal., Rational truncation of an RNA aptamer to prostate-specific membraneantigen using computational structural modeling, Nucleic Acid. Ther. 21(2011) 299e314.

[135] A. Bini, M. Mascini, M. Mascini, A.P. Turner, Selection of thrombin-bindingaptamers by using computational approach for aptasensor application, Bio-sens. Bioelectron. 26 (2011) 4411e4416.

[136] S. Wilhelm, A.J. Tavares, Q. Dai, S. Ohta, J. Audet, H.F. Dvorak, et al., in:Analysis of Nanoparticle Delivery to Tumours, vol. 1, 2016, 16014.