DNA and RNA Nanotechnology 2015; 2: 42–52
Mini review Open Access
Martin Panigaj*, Jakob Reiser Aptamer guided delivery of nucleic acid-based nanoparticles
DOI 10.1515/rnan-2015-0005 Evolution of Ligands by Exponential enrichment) [4,5]. Received July 15, 2015; accepted October 3, 2015 Nucleic acid-based aptamers are especially well suited Abstract: Targeted delivery of bioactive compounds is a for the delivery of nucleic acid-based therapeutics. Any key part of successful therapies. In this context, nucleic nucleic acid with therapeutic potential can be linked acid and protein-based aptamers have been shown to to an aptamer sequence [6], resulting in a bivalent bind therapeutically relevant targets including receptors. molecule endowed with a targeting aptamer moiety and In the last decade, nucleic acid-based therapeutics a functional RNA/DNA moiety like a small interfering coupled to aptamers have emerged as a viable strategy for RNA (siRNA), a micro RNA (miRNA), a miRNA antagonist cell specific delivery. Additionally, recent developments (antimiR), deoxyribozymes (DNAzymes), etc. In addition in nucleic acid nanotechnology offer an abundance of to the specific binding, many aptamers upon receptor possibilities to rationally design aptamer targeted RNA recognition elicit antagonistic or agonistic responses that, or DNA nanoparticles involving combinatorial use of in combination with conjugated functional nucleic acids various intrinsic functionalities. Although a host of issues have the potential of synergism. Since the first report including stability, safety and intracellular trafficking describing an aptamer-siRNA delivery approach in 2006 remain to be addressed, aptamers as simple functional many functional RNAs and DNAs conjugated to aptamer chimeras or as parts of multifunctional self-assembled sequences have been tested in vitro and in vivo [7-9]. RNA/DNA nanostructures hold great potential for clinical Recent progress in the structure predictions of nucleic applications. acids has led to the implementation of protocols for self-assembly of RNA/DNA nanoparticles [10-14]. Such Keywords: Targeted delivery, aptamers, functional structures may provide scaffolds for simultaneous delivery chimeras, nucleic acid-based therapeutics, RNA/DNA of various functionalities, including aptamers as well as nanoparticles fluorescent dyes, chemotherapeutics and/ or proteins. This mini review highlights recent efforts on the use of nucleic acid aptamers as targeting moieties for nucleic 1 Introduction acid-based nanoparticles.
Nucleic acid-based aptamers have gained prominence 2 Aptamers as functional chimeras as a promising platform for targeted drug delivery due to their ability to serve either as a therapeutic agent or as 2.1 Targeting the RNA interference (RNAi) an evolvable targeting modality [1-3]. Nucleic acid-based aptamers consist of in vitro selected oligonucleotides pathway mediated by nucleic acid aptamers involving libraries of up to 1016 randomly synthesized Knock down of gene expression provides a promising tool sequences through a method termed SELEX (Systematic in the context of nucleic-acid based therapies [15]. Uptake of silencing RNAs by cells is greatly facilitated by coupling them to aptamers. While cellular uptake of an siRNA linked *Corresponding author: Martin Panigaj, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, to an aptamer leads to silencing of one specific gene, miRNA Moyzesova 11, 040 01 Košice, Slovakia, e-mail: martin.panigaj@ allows simultaneous repression of multiple genes. upjs.sk In a recent proof-of-concept study for in vivo delivery Jakob Reiser, Division of Cellular and Gene Therapies, FDA/CBER, of aptamer (Apt) -miRNA conjugates the integration of 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA three functionalities in one simple RNA construct was
© 2015 Martin Panigaj, Jakob Reiser, published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Aptamer guided delivery of nucleic acid-based nanoparticles 43 demonstrated. In this construct the antagonistic aptamer the complementary stick sequence was base paired with provided receptor specificity while the miRNA moiety the precursor of miR-126 (pre-miR-126). All three constructs resulted in the silencing of gene expression. In one such were internalized by endothelial cells, but only treatment approach Esposito et al. linked the GL21.T aptamer that is with aptamer-pre-miR-126 resulted in the reduction of specific to Axl, a receptor tyrosine kinase (an oncogene the VCAM-1 target and an increase in angiogenesis. Also, overexpressed in several human cancers), to the tumor there were differences in the response of tumor cell lines suppressor let-7g miRNA, resulting in the GL21.T-let chimera to treatment with Ch3. It is unclear why no biological (Fig. 1A) [16]. Application of the GL21.T-let chimera to mice function was detected with the other two chimeras. bearing A549 (Axl+) tumors downregulated let-7g target Zhou et al. selected an aptamer targeting human genes leading to apoptosis, decreased cell proliferation, B-cell activating factor receptor (BAFF-R) to deliver siRNAs and a reduction in tumor size. Using different conjugation to non-Hodgkin’s lymphoma cells [22]. Activation of strategies and a combination of additional aptamers BAFF-R upregulates pathways leading to enhanced B-cell (e.g., a prostate specific membrane antigen aptamer) and proliferation, but neither aptamers R1 or R14 activated miRNA sequences (e.g., miR-16) the authors demonstrated BAFF-R. Down-regulation of STAT3 was previously the versatility of their approach. demonstrated to inhibit growth of lymphoma tumor cells. The aberrant regulation of certain miRNAs during To deliver siRNAs corresponding to STAT3 two pairs of Apt- malignant transformation makes them potential siRNA chimeras were designed. In the first pair, the sense therapeutic targets for inhibition using antimiRs [17]. or antisense strands were linked to an aptamer involving AntimiRs are synthetic oligonucleotides that base pair an eight-nucleotide linker (Fig. 1D). In the second pair with corresponding miRNAs. Such miRNAs complexed of chimeras, the siRNAs were connected to the aptamer with antimiRs do not bind target mRNAs resulting in an through stick sequences (Fig. 1D). All chimeras led to the increase in the expression of the target gene repressed silencing of STAT3 expression, albeit down-regulation by the miRNA [18]. Recently, Pofahl et al. reported on the mediated by Apt-linker-siRNAs was more profound than delivery of an antimiR, targeting the miR-21, through the using Apt-stick-siRNA chimeras. nucleolin-binding aptamer (AS1411) [19]. Blocking of a cellular receptor with an aptamer In a follow up study the de Franciscis’ group used the represents a plausible strategy to prevent binding and entry GL21.T aptamer to deliver RNA-based antimiRs inhibiting of viruses into host cell. Similarly, binding of aptamers to the cancer progression-associated miRNA miR-222 (Fig. viral glycoproteins decreases virus attachment to target 1B) [20]. Treatment of Axl or platelet-derived growth factor membrane proteins [23,24]. The first approach also offers receptor β expressing cells with the GL21.T-222 or Gin4.T-222 the potential to bolster antiviral effects through aptamer conjugates resulted in the decrease of target miR-222 levels delivery of siRNAs targeting viral and virus-required host and an increase of the miR-222 target proteins. Again, as in genes. previous work, the authors observed a synergistic effect of In two recent studies Zhou et al. investigated functional the GL21.T-222 conjugate on the reduction of cell migration in vivo delivery of anti-HIV-1 siRNAs via RNA aptamers and an increase in the sensitivity to temozolomide induced targeting HIV-1 glycoprotein gp120 or human CCR5 (C-C cell death. Interestingly, when the authors intended to chemokine receptor type 5). In the first study, an aptamer enhance the antagonizing potential using two antimiR- targeting HIV-1 glycoprotein gp120 and a siRNA were joined 222 moieties connected in tandem, no additive effect was via a “sticky bridge” [25]. Complementary sticky bridge detected. In contrast, employing an aptamer with two strands were connected to the 3′-end of the aptamer and different antimiRs (antimiR-222 and antimiR-10b) led to to one of the antisense or sense siRNA sequences using the reduction of the two respective miRNAs and increased a carbon linker. To prevent the creation of viral escape target protein levels. mutants, both virus-encoded and cellular transcripts In a related study three different strategies for were targeted including the viral transcripts encoding conjugating miRNAs (miR-126) to a transferrin receptor HIV-1 tat and rev, and transcripts encoding CD4 that is aptamer (TfRA) were tested (Fig. 1C) [21]. In the first study, required for HIV-1 entry and transportin 3 (TNPO3) that is a chimera (Ch1) consisting of the mature miR-126-5p required for viral integration. The intravenous injection covalently linked to TfRA and base-paired to miR-126-3p of an anti-gp120 Apt-siRNAs mixture to humanized mice was used. For the Ch2 chimera used in the second study, downregulated expression of all three genes targeted. the TfRA contained a “stick” sequence complementary to Most of the mice treated had undetectable viral loads up a stick part linked to the mature miR-126-5p annealed to to three weeks after the final treatment. Moreover, viral miR-126-3p. The third chimera (Ch3) was similar to Ch2 but rebounds observed three months after the end of the first 44 M. Panigaj, J. Reiser Figure 1
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Figure 1: Depiction of selected chimeric aptamers discussed in this review. Panels A and B show an aptamer (GL21.T) targeting Axl, a recep- tor tyrosine kinase, linked to let-7g miRNA or to an antimiR recognizing miR-222, respectively. Panel C displays three separate conjugates involving both the 3p and the 5p forms of miR-126 as well as the precursor pre-miRNA 126 linked to a transferrin receptor (TfR) aptamer. Panel D, upper graph: An aptamer (R1) targeting the human B-cell activating factor receptor linked through a poly U linker to the sense or antisense strands of the STAT3 siRNA. Panel D, lower graph: The sense or antisense strands of the siRNA connected to the RNA aptamer through a carbon linker-stick sequence. Panel E: A DNA aptamer against Mucin 1 conjugated to anti GFP or anti Bcl-2 siRNAs in a multivalent comb-like shape. Antisense siRNA strands coupled through disulfide bonds are base paired to the DNA aptamer- siRNA sense strand con- jugate. Panel F: A Dimeric 4-1BB receptor-specific aptamer base-paired to a VEGF aptamer. Panel G shows a short G-C rich double stranded DNA tail-antitail sequence conjugated through a disulfide linker to a NF-κB self-complementary decoy sequence. Aptamer guided delivery of nucleic acid-based nanoparticles 45 treatment were fully suppressed by re-injection of the who annealed a nucleolin binding DNA aptamer (AS1411) Apt-siRNA conjugates. The administration of anti-gp120 to a survivin specific DNAzyme [30]. Nucleolin is able to Apt-siRNAs mixtures also inhibited HIV-1 mediated CD4+ rapidly translocate from the cell surface to the nuclei of T-cell depletion. In their subsequent work Zhou et al. cells which makes it an ideal transport vehicle to reach selected an RNA aptamer (G-3) targeting the human CCR5 the nucleus. Survivin is a member of a protein family that protein that alone decreased R5 HIV-1 virus replication promotes survival and proliferation of cancer cells [31]. when applied prior or post infection with HIV-1 virus in a Surprisingly, the WERI-Rb1 cell line with the internalized population of PBMC-CD4+ cells. However, suppression of chimeric conjugate showed decreases in survivin mRNA HIV-1 replication was stronger when G-3-TNPO3 siRNA was levels and cell proliferation similar to cells treated with applied [26]. the aptamer alone. Because the nucleolin aptamer by Recent results using a humanized murine model itself also triggered functional responses it is tempting to suggest that hydroxyethyl cellulose gel-formulated speculate that its binding to nucleolin may have down- Apt-siRNA chimeras may also be used to prevent HIV regulated survivin expression. infection in the female genital tract [27]. Mice that received CD4+ specific aptamers with covalently linked siRNAs 2.3 Aptamer-mediated delivery of splice- corresponding either to the host CCR5 gene or the viral switching oligonucleotides gag/vif sequences up to four days before intravaginal exposure to virus showed no evidence of infection. In Splice-switching oligonucleotides (SSOs) are single- the following 10 weeks no p24 antigen or viral RNA stranded oligonucleotides that target nuclear pre-mRNA was detected in their plasma and the number of CD4+ molecules to alter mRNA splicing resulting in alternative cells remained unchanged. Even administration of CD4 protein isoforms [32]. Conjugation of SSOs to an aptamer aptamer-gag/vif siRNAs for just four hours post exposure whose target protein (e.g. nucleolin) is translocated to the protected some mice completely. nucleus may enhance the potential therapeutic utility of A relatively simple idea of how to improve the SSOs. intracellular uptake of siRNAs due to the increased In a proof-of-principle experiment, Kotula et al. used chances of contact between aptamers and receptors was a prostate cancer cell line expressing a luciferase reporter outlined by Yoo et al. who “chained” a DNA aptamer construct with a premature stop codon. When treated with against Mucin 1 and anti GFP or Bcl-2 siRNA conjugates the nucleolin Apt-luciferase SSO chimera the percentage in a multivalent comb-like structure [28]. The backbone of of repaired mRNA increased by 13% over the baseline as the rod-like nanoparticle was created by linear connection seen in untreated cells but only by 5% compared with cells of antisense siRNA strands via thiol-maleimide coupling. treated with the luciferase SSOs only. One major advantage Subsequently, multiple copies of DNA aptamers-sense of SSO-based therapies is that only a small percentage of siRNA conjugates were annealed to antisense siRNA chains repaired pre-mRNA often times is sufficient to generate a (Fig. 1E). Compared to monomeric and dimeric Apt-siRNA, phenotypic effect [33]. multimeric Apt-siRNA labeled with the red fluorescent dye POPO-3 in the context of MUC1-positive human MCF-7 2.4 Bispecific aptamer conjugates breast cancer cells produced the strongest fluorescence signal. However, despite efficient internalization no Linking various cell-specific aptamers to a DNA apparent GFP knock-down was detected, possibly scaffold provides an appealing strategy to direct cell- pointing to poor endosomal escape. cell interactions [34]. For example, bispecific aptamer conjugates have the potential to cross-link tumor cells 2.2 Aptamer-mediated delivery of with immune cells and to activate the cognate receptors. catalytically active nucleic acids The 4-1BB receptor plays a crucial role in the survival and expansion of activated CD8+ T cells and their differentiation DNAzymes are single stranded DNA molecules that are into memory cells. The lack of the 4-1BB ligand in tumors catalytically active on nucleic acid substrates catalyzing inspired Gilboa’s group to conjugate the agonistic 4-1BB a variety of reactions like RNA cleavage, ligation, DNA aptamer to an aptamer targeting VEGF (Fig. 1F) [35]. phosphorylation etc. Several studies have already examined VEGF is secreted into the stroma of many tumors, which the ability of DNAzymes to destroy specific mRNAs [29]. makes it a versatile target. Vaccination and co-treatment The first aptamer-mediated targeted delivery of a with the VEGF4-1BB aptamer effectively inhibited tumor DNAzyme was recently described by Subramanian et al. growth while conjugates alone or vaccination had a 46 M. Panigaj, J. Reiser minimal effect. Vaccination with a mixture of VEGF and components can be associated with nanoparticles such as 4-1BB aptamers also had no effect. Similar outcomes were fluorophores, chemotherapeutics, etc. [39-41]. observed in the context of four unrelated tumor models. The origin of RNA nanotechnology dates back to 1998, but surprisingly, very few groups reported targeted 2.5 Aptamer-mediated delivery of decoys delivery of self-assembled RNA nanoparticles through aptamers [42]. In pioneering work Guo et al. treated CD4- Decoys are synthetic, double-stranded oligonucleotides overexpressing T cells with a RNA nanoparticle consisting that selectively inhibit the transcriptional activity of DNA of a dimer of the packaging RNA (pRNA) derived from binding proteins by competing for binding to their specific the DNA-packaging motor of bacteriophage phi29, a CD4 target sequences present in promoters and enhancers [36]. specific aptamer and a siRNA complementary to survivin Cell specific delivery of decoys through aptamers mRNA (Fig. 2A) [43]. Although, survivin mRNA or protein was recently demonstrated by Porciani et al. [37]. In levels were not investigated, the decrease in cell viability their model system they evaluated the ability of an RNA observed was due to the targeted delivery of survivin aptamer binding to human transferrin receptor to deliver siRNA, because incubation of cells with survivin pRNA- a decoy designed to mimic the κB consensus promoter siRNA monomers alone had no effect on cell viability. sequence recognized by NF-κB. Various stimuli such as Since the intermolecular binding domain and the double- carcinogenesis, inflammatory agents, or chemotherapeutic stranded helical domain at the 5′ and 3′ ends of pRNA fold drugs activate NF-κB resulting in its translocation from the independently exchange of the end helical region for an cytoplasm to the nucleus where it promotes transcription aptamer or siRNA did not affect the pRNA structure provided of antiapoptotic genes. To achieve a synergistic effect, that the two strands were base-paired. The left- and right- Porciani et al. elongated the aptamer with a doxorubicine hand loops of the interlocking domain were shown to pair (Dox) binding G-C rich double stranded DNA tail that with other monomers to form multimeric pRNAs [12]. More hybridized to an antitail sequence conjugated through recently, Hu et al. used this feature to create dimeric pRNAs a disulfide linker to a NF- κB self-complementary decoy (FRS-NPs) where the helical domain of one monomer sequence (Fig. 1G). Internalization of the Apt-NF-κB was replaced with the FB4 aptamer directed against the decoy by pancreatic carcinoma MIA PaCa-2 cells led to mouse transferrin receptor and the helical domain of the the reduction of NF-κB translocation from the cytoplasm second monomer with an siRNA targeting ICAM-1 mRNA. to the nucleus upon TNF-α stimulation. Subsequently, Treatment of an in vitro inflammatory cell model with simultaneous delivery of Dox and inhibition of NF-κB FRS-NPs reversed the increase of ICAM-1 expression and resulted in higher cytotoxicity than using an aptamer-Dox blocked the adhesion of monocytes [44]. without decoy. In a recent study Afonin et al. designed artificial multifunctional RNA nanoparticles and examined their capacity to silence multiple HIV-1 genes using siRNAs, to 3 Aptamers as parts of multifunc- deliver newly developed RNA-DNA hybrids into cells and tional self-assembled RNA/DNA to target cells using aptamers [41]. Hexameric nanorings allowed combinatorial embedment of up to 6 different nanostructures functionalities, including aptamers and siRNAs, base- paired to the ring scaffold (Fig. 2B). To demonstrate that DNA and RNA nanoparticles are programmable engineered nanorings can be targeted to specific cells, particles 3D nanoscaffolds that benefit from the unique ability of containing up to five copies of the J18 RNA aptamer binding nucleic acids to form canonical and non-canonical base to the human epidermal growth factor receptor (EGFR) were pairings leading to a diverse set of structural motifs. assembled [45]. One biotinylated oligonucleotide that was The simplicity of their primary structure allows self- part of the nanoring structure served as a coupling system assembly into compact and stable structures with precise allowing fluorescent visualisation through streptavidin control over the size, geometry, and composition of the conjugated to phycoerythrin. Nanorings with higher final nanoparticle [38]. Research carried out in recent numbers of aptamers per nanoparticle provided a higher years showed that DNA and RNA nanoparticles are binding affinity to target cells. The internalization and suitable as scaffolds for the delivery of various modules functional effects of siRNAs bound to aptamer- nanorings carrying different functional properties like aptamers were not studied. and antisense oligonucleotides. In addition to intrinsic Although DNA lacks the functional versatility of nucleic acid-based functional moieties, other chemical RNA, self-assembled DNA nanoparticles have been Figure 2 Aptamer guided delivery of nucleic acid-based nanoparticles 47
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Figure 2: Aptamers as parts of multifunctional self-assembled RNA/DNA nanostructures. Panel A: Dimeric pRNA formed by interaction of the left- and right-hand loops of the interlocking domain. Panel B: Hexameric nanoring with five J18 aptamers and one biotinylated DNA oligonucleotide allowing labeling using a streptavidin-phycoerythrin conjugate. Panel C: Aptamer tethered DNA nanotrains consisting of aptamers targeting protein tyrosine kinase 7 (sgc8) or nucleolin (AS1411). The aptamers are base-paired to linear repeating DNA building blocks that allow Dox loading. Panel D: A hexagonal barrel shaped nanorobot consisting of two halves (orange and blue structures) linked by single-stranded scaffold hinges (not shown). The opened barrel with exposed payload is shown. In the absence of the aptamer target, the nanorobot is locked by a DNA lock consisting of the two DNA aptamers base-paired with the partially complementary strands on the opposite domain. successfully used as a simple scaffold with the capacity The DNA origami technique provides a powerful to bind to the folate receptor and to display antisense DNA method to create custom DNA devices by folding multi- oligonucleotides or to deliver siRNAs [46,47]. Moreover, kilobase ssDNA scaffolds via interaction with “staple” DNA scaffolds are suitable to deliver chemotherapeutics oligonucleotides [50]. Besides building mere static intercalated into its duplex structure as shown by Tan’s structures, several groups explored the nanomechanical group that created the so called Apt-tethered DNA properties of DNA devices made of DNA origami regarding nanotrains (Apt-ntr) consisting of the sgc8 or AS1411 their ability to dynamically react on external stimuli [51]. aptamers followed by a long linear dsDNA nanostructure Cell-specific delivery and logical release of cargo based on (Fig. 2C) [48]. Application of Apt-ntr loaded with Dox in the specificity of the aptamers used was demonstrated by a mouse xenograft tumor model reduced tumor volumes the Church group who created a nanorobot in the form of a more efficiently than free Dox and led to prolonged hexagonal barrel consisting of two halves linked together survival of treated mice. Furthermore, mice treated with by single-stranded scaffold hinges (Fig. 2D) [52]. Inspired Dox delivered in a targeted fashion experienced less by Andersen et al., this barrel was locked by two DNA weight loss compared to mice treated with “free” Dox, aptamers present on one of the half-barrel domains and indicating that targeted delivery reduced side effects. In was base-paired to a partially complementary strand on a parallel study Tan and coworkers photo-cross-linked the opposite domain [53]. After simultaneous recognition 5’ acrydite- modified oligonucleotides to form spherical of target proteins (protein keys) by the two aptamers, the DNA nanoparticles designed to deliver the MDR1-specific aptamer lock duplexes dissociated, and the nanorobot antisense oligonucleotides and Dox to kill drug resistant exposed its cargo hidden inside of the barrel. Payloads myelogenous leukemia K562/D cells [49]. conjugated to the 5′ end of ssDNA oligonucleotides 48 M. Panigaj, J. Reiser involved up to 12 complementary 3′ extended staple selection methods such as cell-internalization SELEX strands. To prove the functionality and robustness of the have been developed [55,56]. It appears that the aptamer-encoded logic gating approach, six different current strategies employed to link aptamers with a robots were designed using pairwise combinations of functional module preserves folding and consequently aptamer locks from a set of three aptamer sequences: 41t, aptamer target affinity [16,20,22,25]. Furthermore, TE17, and sgc8c. Barrels loaded with fluorescently labeled the computational design of self-assembling nucleic Fab’ fragments against human leukocyte antigens emitted acid nanoparticles allows assembling of individual a fluorescent signal only when antibody fragments were functional parts on the scaffolds while preserving their uncovered from the barrel interior after the two protein proper folding [57]. keys on the cell surface were matched to an aptamer pair. In mixed cell populations the robots displaying the proper 4.2 Sensitivity to nucleases aptamer locks bound almost exclusively to the target NKL cells and detected one target cell in a background The sensitivity of nucleic acids to nucleases in of 106 receptor negative cells. In addition to cell labeling, vivo complicates their therapeutic use. However, nanorobots carrying a combination of antibodies incorporation of chemically modified nucleotides such triggered physiological response in targeted cells by as 2’-F pyrimidines and 2’-Ο Me nucleotides or the use of inducing growth arrest in leukemic cells or promoting phosphorothioate internucleotide linkages significantly T-cell activation. extends survival of nucleic acids without affecting their The prevalent strategy for creating DNA nanoparticles functionality [58-60]. utilizes Watson-Crick base-pairing between individual The self-assembled DNA/RNA nanocarriers adopt DNA building blocks. In an alternative approach Zhou conformations that substantially differ from naturally et al. created densely packed DNA particles with built-in occurring nucleic acids [61]. It is thus questionable to multifunctional moieties that self-assembled through what extent a conclusion about their stability based on the liquid crystallization to monodisperse structures knowledge of their building blocks can be drawn. To date without reliance on Watson-Crick base-pairing [54]. several groups have reported that 3D DNA nanostructures DNA concatemers consisting of multiple aptamer (sgc8) of various shapes maintained their structural integrity sequences and Dox loading were generated using rolling up to 48 hours in the cytoplasm or lysosomes or when circle replication. The so-called Nanoflowers (NFs) exposed to cell lysates, or nucleases [62-65]. The specifically recognized target HeLa and CEM cells and relationship between stability and shape has shown that subsequent treatment with Dox loaded NFs or free Dox led triangular nanostructures tend to be more fragile than to a similar decrease in cell viability. However, Dox-NFs tetrahedral structures [66]. The degradation kinetics of reduced the viability of control Ramos cells only modestly DNA box origami in serum have been investigated in real compared to free Dox. time with nanometer resolution. High-speed atomic force microscopy revealed that the half lives of 3D DNA box origami structures in 0.1 % serum is 62 ± 26 s and that 4 Issues to be resolved destruction started with a rapid height collapse phase and a slow degradation phase that lasted from several minutes Delivery of nucleic acid-based therapeutics through to half an hour [67]. aptamers poses several challenges including but not limited to the structural design, in vivo stability, 4.3 Clearance immunogenicity, cellular uptake and endosomal escape.
Besides degradation by nucleases, circulation of nucleic 4.1 Aptamer design acid-based therapeutics in the blood stream is challenged by their removal through size dependent extravasation The selection of an optimal aptamer that is highly and renal clearance. In addition to cellular factors, specific for its target in the native microenvironment nanoparticle size, shape, and distribution of ligands (cell membrane) and that is endocytosed upon binding on the nanoparticle were shown to influence the rate of and released into the cytoplasm, is quite challenging. cellular uptake [68]. To approach this issue modified cell-based aptamer Aptamer guided delivery of nucleic acid-based nanoparticles 49
4.4 Immune responses [70]. Interestingly, 2’-F and 2’-Ο Me modified nucleotides do not promote a defense reaction [71]. On the other hand Following internalization nucleic-acid therapeutics are the immunomodulatory properties of nucleic acids can be subject to the cellular innate immune surveillance system. exploited to trigger innate immunity responses [72-74]. Nucleic acids in the endolysosomal lumen are screened by nucleic acid-sensing Toll like receptors (TLRs) based on 4.5 Trafficking their structure and sequence (Fig. 3) [69]. Furthermore, many nonimmune cells (epithelial cells, fibroblasts) that The trafficking of an internalized aptamer bound to a do not express TLRs are able to trigger innate immune receptor can be complex. For example, the endocytosed responses by a second line of sensors localized in the payload can end up being degraded in lysosomes, or cytosol, suggesting that not all experimental settings may exported out of the cell (Fig. 3). Alternatively, it can revealFigure the immunogenic3 potential of nanoparticle (Fig. 3) escape from the endosome. The fate of the aptamer-
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> pathways leading to undesired outcomes > pathway leading to desired outcome Figure 3: Trafficking of internalized nucleic-acid therapeutics. The aptamer RNA/DNA nanoparticle-receptor complex is internalized and distributed in a number of ways. Undesirable outcomes are recycling of the particle back to the cell surface, degradation in lysosomes or triggering of cellular defense reactions. Nucleic acids activate the innate immune system in the endosomal compartment through the TLRs that recognize ssRNA or dsRNA and unmethylated cytosine-guanosine (CpG) DNA motifs. Subsequent signalling cascades induce transcrip- tion of type I interferon (IFN) genes and genes encoding other inflammatory cytokines. Foreign RNA that reaches the cytoplasm is sensed by the cytosolic surveillance system, represented most notably by the retinoic acid inducible gene-I (RIG-I), 2‘-5- oligoadenylate synthetase (OAS), RNase L and dsRNA-depended protein kinase R (PKR). RIG-I recognizes a triphosphate group at the 5’ end of the RNA strands or short RNAs generated by RNase L. When bound to dsRNA, OAS converts ATP to 2’- 5’-linked oligoadenylates that activate degradation of ssRNAs through RNase L. Binding of PKR to the dsRNA leads to suppression of translation. Ultimately, the transcription of type I IFN genes and pro- inflammatory cytokine genes is activated. For simplicity, many proteins involved in downstream signaling are omitted as well as proteins involved in cytosolic DNA sensing such as the absent in melanoma 2 (AIM2) and STING proteins. Since the cellular defense is triggered by the recognition of the sequence/pattern of nucleic acids that are specific for particular infectious agents, it is challenging to extrapolate these findings to nucleic acid-based nanoparticles. In addition, the structure of viral genomes typically cannot be used for the design of therapeutically useful nanoparticles. 50 M. Panigaj, J. Reiser
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