CHEMMEDCHEM REVIEWS DOI: 10.1002/cmdc.201402163

Aptamers as Drug Delivery Vehicles Sven Kruspe, Florian Mittelberger, Kristina Szameit, and Ulrich Hahn*[a] Dedicated to Wolfram Saenger on the occasion of his 75th birthday.

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CHEMMEDCHEM REVIEWS The benefits of directed and selective therapy for systemic treatment are reasons for increased interest in exploiting aptamers for cell-specific drug delivery. Nucleic acid based pharmaceuticals represent an interesting and novel tool to counter human diseases. Combining inhibitory potential and cargo transfer upon internalization, nanocarriers as well as various therapeutics including siRNAs, chemotherapeutics, photosensitizers, or proteins can be imported via these synthetic nucleic acids. However, widespread clinical application is still ham-

Introduction Since their first mention in 1990[1, 2] aptamers have evolved to sophisticated tools in molecular biology,[3, 4] nanotechnology,[5] and medicinal sciences.[6, 7] Aptamers are short single-stranded oligonucleotides (ONTs) with recognition properties for certain target molecules due to their unique three-dimensional folding. They show high affinity and specificity for target molecules such as proteins,[8, 9] peptides,[10] metal ions,[10] small molecules,[11] and even whole cells.[12, 13] Aptamers possess substantial advantages over antibodies, such as little to no immunogenicity and toxicity,[14, 15] longer shelf-life, lower production costs, and low batch-to-batch variation. They are typically obtained from a selection method termed SELEX (systematic evolution of ligands by exponential enrichment; Figure 1). Aptamers that bind to cell-surface proteins, such as overexpressed receptors on cancer cells, can be used as delivery vehicles to convey cargos into certain cell types for diagnostic or therapeutic purposes. Upon binding to a surface protein they

Figure 1. The SELEX process. Generation of aptamers is performed by the iterative selection of binders from a starting pool. The starting pool contains RNA or DNA oligonucleotides comprising constant terminal regions for primer annealing and a randomized central region (typically 20–50 nt). Upon exposure to the target of interest (1) binders are separated from the nonbinding fraction (2). After elution from the target molecule (3) binders are amplified by RT-PCR (RNA) or PCR (DNA) (4). Single-strand synthesis leads to an enriched oligonucleotide pool (5), which is repeatedly subjected to steps 1–5. Aptamers are elucidated by sequence analysis of the final pool.

[a] S. Kruspe, F. Mittelberger, K. Szameit, Prof. Dr. U. Hahn Institut fr Biochemie und Molekularbiologie, Universitt Hamburg Martin-Luther-King Platz 6, 20146 Hamburg (Germany) E-mail: [email protected]

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www.chemmedchem.org pered by obstacles that must be overcome. In this review, we give an overview of applications and recent advances in aptamer-mediated drug delivery. We also introduce prominent selection methods as well as useful approaches in choice of drug and conjugation method. We discuss the challenges that need to be considered and present strategies that have been applied to achieve intracellular delivery of effectors transported by readily internalized aptamers.

use endocytic internalization processes to enter the cell. During the past decade numerous aptamers have been applied in such drug delivery approaches. Several challenges in aptamer-mediated drug delivery must be overcome, the most important of which is degradation by nucleases present in the serum, particularly for RNA aptamers. Chemical modifications of the sugar unit, the phosphate backbone, or the nucleobases are used to solve this problem and can be introduced either post-selectively or in the synthesis steps of SELEX. Substitution of the 2’-hydroxy group in RNA by fluorine (2’F), methoxy (2’-OMe), or O-methoxyethyl (2’-MOE) increases resistance to endonuclease activity. Alternatively, locked nucleic acids (LNAs) containing 2’-O-, 4’-C-bridges can be applied. This modification also enhances structural stability by locking the 3’-endo conformation of ribose.[16] The addition of a 3’-inverted thymidine (3’-invT) supports resistance against exonucleases. Also, oxygen in the phosphate backbone can be replaced with sulfur, resulting in phosphothioate bonds, which are less susceptible to degradation as a consequence of binding to serum proteins. Enantiomers of natural RNA (l-RNA or spiegelmers) represent another smart approach to ensure nuclease resistance. Modifications that decrease renal clearance of small molecules imply conjugation of cholesterol or polyethylene glycol (PEG) to the aptamer.[7, 17] Despite these considerations, cell-surface binding aptamers might exhibit limited success for therapeutic applications due to insufficient uptake or cargo release. Therefore, the appropriate choice of target is essential for targeted delivery. The first section of this review deals with prominent aptamers that have been applied in delivery studies, their targets, and the various strategies by which they were selected. In the second section we point out considerable prerequisites that account for successful aptamer–drug conjugation and introduce common effector molecules, focusing on advancements that have taken place over the past few years.

Selection of Cell-Targeting Aptamers There are several routes to select aptamers for a given target associated with a malignant cell type. The most straightforward method involves immobilization of the desired molecule on a column or beads. This approach has the advantage of the target being in pure form; however, it also bears the risk of selecting aptamers that do not bind the target in its native state. ChemMedChem 0000, 00, 1 – 15

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CHEMMEDCHEM REVIEWS This disadvantage can be circumvented by conducting the selection with whole cells (cell-SELEX).[18] This approach requires more expertise and may result in the selection of aptamers for cell-surface molecules other than the desired target. Cell-based SELEX provides several advancements such as blind SELEX (seSven Kruspe studied chemistry and obtained his Diplom from Hamburg University in 2010. He has since been carrying out his PhD thesis research in the research group of Professor Hahn in the Department of Chemistry at Hamburg University in the field of aptamer-mediated drug delivery.

Florian Mittelberger studied biology at Hamburg University and received his master’s degree in plant molecular biology in 2010. He started his PhD work in Professor Hahn’s research group in the Department of Chemistry at Hamburg University in 2011, where he works on selection and characterization of RNA aptamers.

Kristina Szameit studied biochemistry at Hamburg University and received her Diplom in 2011. Since then, she has been working toward her PhD in Professor Hahn’s group in the Department of Chemistry at Hamburg University. Her research is focused on investigation of aptamer internalization and structure determination.

Ulrich Hahn received his PhD at the Max Planck Institute for Experimental Medicine and Georg August University at Gçttingen in 1980. He was part of the research group of Wolfram Saenger (1982–1993, Free University of Berlin), and lecturer at the University of Lbeck (1993–1994). He became Full Professor in 1994 at Leipzig University and in 2002 at Hamburg University. His main topics of scientific research are currently molecular enzymology and RNA biochemistry (aptamers).

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www.chemmedchem.org lection for an unknown target presented on a certain malignant cell type) or internalization SELEX (exclusive selection of aptamers to adequately internalized receptors by high salt washing steps or trypsinization of surface proteins after initial incubation).[19–22] Prostate-specific membrane antigen (PSMA) A9 and A10 are 2’-fluoro-2’-deoxypyrimidine (2’-F-Py) RNA aptamers that were selected for binding to the extracellular domain of PSMA (xPSM), using immobilized protein.[9] Prostatespecific membrane antigen (PSMA) is a transmembrane protein ubiquitously associated with prostate cancer and abundantly expressed by neovascular tissue of solid tumors.[23] In healthy prostate tissue, the expression is mostly confined to a spliced form of PSMA, lacking the transmembrane domain (PSM’).[24] Membrane-bound PSMA is readily internalized in an at least partly clathrin-dependent manner.[25, 26] Using 2’-F-Pys not only has the advantage of greatly increasing the resistance of the RNA against abundant serum nucleases,[27] but also increases the thermal stability of RNAs. A NAALADase assay was performed to test xPSM activity before selection and ensure native protein folding for the in vitro SELEX.[28] The protein was immobilized on magnetic beads and incubated with pool RNA at 37 8C. Incubation and washing steps were performed in HBSMC buffer [HEPES-buffered saline (pH 7.4), 1 mm MgCl2, and 1 mm CaCl2]. The two selected aptamers showed inhibitory activity toward PSMA, as confirmed by the same assay used to monitor protein integrity prior to SELEX. Whereas A9 inhibits PSMA noncompetitively with a Ki value of 2 nm, A10 shows competitive inhibition with Ki = 11.9 nm.[9] A shortened form of A10 was shown to bind to PSMA-expressing LNCaP prostate cancer cells. Later experiments showed that functionalized A10 could be used for in vivo studies and was effective even with systemic administration.[29] Rational truncation yielded different variants of A9 (e.g., A9g and A9L)[30] that were applied in delivery approaches.[31] Mucin Epithelial mucin MUC1, regularly present in normal glandular epithelial cells, is a well-characterized marker for adenocarcinomas including breast, lung, and colon cancers.[32, 33] In malignant cells the expression level is strongly elevated, and MUC1 shows an aberrant glycosylation pattern.[34] The MUC1 DNA aptamer was selected for the immobilized nine-residue peptide APDTRPAPG corresponding to a highly immunogenic variable tandem repeat (VTR).[35] This epitope is only accessible in the mucin of breast cancer, but not of normal breast epithelial cells.[36] Two selections were performed: one using a high salt buffer (200 mm NaHCO3, 500 mm NaCl, pH 7.4), the other using a buffer corresponding to physiological conditions (100 mm NaCl, 5 mm MgCl2, pH 7.2). The best binders of both selections shared an identical sequence. However, other aptamers selected under high salt conditions showed no binding to the target under physiological conditions, highlighting the importance of choosing selecChemMedChem 0000, 00, 1 – 15

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CHEMMEDCHEM REVIEWS tion conditions that closely resemble those of the desired application.[35] The APDTRPAPG peptide used for the SELEX had been previously shown to be unstructured in solution, rendering it a rather unusual target for aptamer selection.[37] However, fluorescence microscopy analysis of MCF-7 breast cancer cells incubated with rhodamine-labeled MUC1–aptamer confirmed binding to mucin on the cell surface. The authors suggested that binding of the aptamer stabilizes the structure of the peptide, allowing it to recognize the exposed VTR of mucin.[35] Nucleolin The nucleolin-specific DNA aptamer AS1411 is an unusual representative, as it has not been obtained by SELEX. This dimeric G-quadruplex-forming aptamer was identified in a study investigating the effects of G-rich ONTs on the growth of tumor cells in culture.[38] Early results indicated the aptamer binding to nucleolin, a multifunctional heterogeneous nuclear ribonucleoprotein (hnRNP) of the RNA binding protein (RBP) family.[38] Nucleolin is an outstanding target for aptamers because of its cellular localization and molecular functions. It was shown to be present in the nucleus, cytoplasm, and cell surface,[39–41] making it accessible to extracellular factors and allowing them to be shuttled to the cytoplasm. Extranuclear localization of nucleolin correlates with differences in its glycosylation patterns.[42, 43] This versatile protein is involved in a wide variety of cellular processes, including rRNA transcription and processing, ribosome assembly and maturation, pre-mRNA metabolism, and cytoplasmic RNA stability.[44–46] High proliferating cancer cells, including breast cancer, lymphocytic leukemia, and prostate carcinoma, show elevated nucleolin levels.[47, 48] Upon binding and internalization, AS1411 impairs the growth of cancer cells (MCF-7), leading to apoptosis. This effect relies, at least in part, on the inhibition of nucleolin binding to the adenineand uracil-rich instability element (ARE) in the 3’-untranslated region (3’-UTR) of bcl-2 mRNA, leading to faster degradation of the transcript.[49] AGC03 is another DNA aptamer that was selected via cellSELEX with the gastric cancer cell line HGC-27. Confocal laser scanning microscopy revealed a nuclear localization of 6-carboxyfluorescein (FAM)-labeled AGC03 after incubation with HGC-27, suggesting nucleolin as a possible target for the aptamer.[50] A counter-selection step with a closely related cell line was employed to gain specificity of the nucleic acid pool for the cell line of interest. In vivo SELEX Mi et al. went one step further by developing an in vivo SELEX protocol.[51] After injecting the starting library of 2’-F-Py RNA into the tail vein of intrahepatic tumor bearing mice, the authors were able to select aptamers that localize and bind their target under in vivo conditions. Subsequently, the liver tumors were harvested, the RNA extracted and amplified for the next selection round. After 14 successive rounds, the enriched pool was sequenced, and families were identified based on the alignment of consensus motifs.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org RNA 14-16, the most dominant sequence motif in the pool, showed high affinity (Kd = 30.8 nm) for aggregate CT26 tumor proteins and only mild affinity for normal colon tissue. RNA 1416 was then used for affinity purification and MS–MS peptide fragment ion matching for identification of the aptamer target. Analysis revealed that RNA 14-16 binds to mouse p68 RNA helicase (Ddx5).[51] Previous studies had shown that p68 is overexpressed and abnormally polyubiquitinated in colon rectal carcinoma.[52] Like nucleolin, p68 is present in the cytoplasm and nucleus and is possibly able to shuttle between the plasma membrane and these compartments. Drug delivery across the blood–brain barrier (BBB) is still one of the biggest technical hurdles faced by scientists and medical practitioners. Not a single commercially available drug has yet been successfully converted from a non-brain-penetrating drug into a substance that crosses the BBB in pharmacologically significant amounts.[53] The BBB consists of a highly impermeable complex of tight junctions and adherent junctions between adjacent endothelial cells. Crossing of the BBB by diffusion is restricted to small lipophilic molecules, whereas for larger or hydrophilic molecules, active transport mechanisms are required.[54] In an attempt to select aptamers that are capable of crossing the BBB, Cheng et al.[55] used in vivo SELEX with a 2’-F-Py RNA library. Brains were harvested after systemic administration of the pool into mice tail veins, and pool RNA was subsequently isolated and re-amplified by RT-PCR. Interestingly, A14, the most prominent aptamer from round 14 to 18, was absent in the 30 clones tested for round 22. A15, the aptamer compiling 50 % of the pool of selection round 22, was also shown to achieve the highest relative enrichment in the brain relative to other abundant aptamer candidates of the same pool. The tissue-specific localization of A15 was verified by qRTPCR of parenchymal cells separated from blood vessels and in situ hybridization experiments with cerebral cortex, striatum, hippocampus, and cerebellum of mice. Flow cytometry and confocal microscopy analysis revealed that A15 was readily internalized into endothelial cells, suggesting transcytosis mechanisms for BBB penetration.

Aptamer–Drug Conjugates General considerations For targeted drug therapy, drug molecules are covalently or noncovalently attached to one or more sites of the aptamer. Aptamers that have been successfully used for drug delivery are listed in Table 1. Various aspects must be considered regarding chemical conjugation. Common cost-efficient conjugations include the linkage of amino- or thiol-modified nucleic acids to drugs, or linker molecules containing carboxylic acid or maleimide. To ensure controlled release, the choice of drug and conjugation method should also be inspired by the sub-localization of the target after its internalization. There are many endocytic pathways, including the well-characterized clathrin-mediated endocytosis (CME), as well as mechanisms independent of clathrin (e.g., caveolae-mediated ChemMedChem 0000, 00, 1 – 15

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Table 1. Aptamers applied in targeted drug delivery. Target

Aptamers

Type

prostate-specific membrane antigen (PSMA)

A10

RNA, 2’-F-Py in vitro SELEX (immobilized extracellular domain on magnetic beads)

SELEX Method

RNA, 2’-F-Py, 3’-invT

protein tyrosine kinase-7 (PTK7)

A10, A10-3.2

RNA, 2’-F-Py

A10-3

RNA, 2’-F-Py

A10-3.2

RNA, 2’-F-Py

A9

RNA, 2’-F-Py

A9L

RNA, 2’-F-Py

STZTI01

DNA

sgc8

DNA

cell-SELEX

sgc8c

epidermal growth factor receptor (EGFR)

E07

CD4

CD4 aptamer clone 9 and clone 12

RNA, 2’-F-Py in vitro SELEX (immobilized recombinant EGFR–Fc fusion protein on magnetic beads) RNA, 2’-F-Py in vitro SELEX (soluble recombinant CD4 antigen immobilized on sepharose beads)

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Cargo Conjugate

Ref.

Application

co-delivery of doxorubicin and docetaxel in PLGA-PEG-NPs chemotherapeutics (docetaxel, paclitaxel) encapsulated in NPs PtIV prodrug to cisplatin encapsulated in PLGA–PEG NPs doxorubicin-loaded superparamagnetic iron oxide NPs (SPIONs) doxorubicin physical conjugate PI3K inhibitor TGX-221 encapsulated in PEG–PCL micelles QD conjugates co-delivery of doxorubicin and shRNA against Bcl-xL loaded in PEI NPs siRNA-chimeras targeting PLK1 or Bcl-2 shRNA-chimeras targeting EEF2 (bivalent aptamer design) shRNA-chimeras targeting DNAPK for sensitization to ionizing radiation miRNAs loaded on PAMAM dendrimers DsiRNA–streptavidin construct co-delivery of doxorubicin and immune-stimulant CpG on PAMAM dendrimers toxin conjugate (gelonin) siRNA-chimeras on PEI coated QDs covalently fixed doxorubicin in dimeric aptamer doxorubicin intercalated in DNA nanostrains chlorin e6 on Au nanorods viral capsid

[56]

in vitro therapy

[57–60]

in vitro therapy

[61]

in vitro therapy

[62]

in vitro therapy and imaging

[63, 64] [65]

in vitro therapy in vitro therapy

[66] [67]

in vitro imaging in vitro and in vivo therapy (xenograft)

Au–Ag nanorods (photothermal therapy) daunorubicin intercalated in the helical strand of sgc8 multivalent aptamer-modified liposomes porous hollow magnetite nanoparticles loaded with doxorubicin silica NPs loaded with doxorubicin DNA origami nanorobot loaded with antibody fragments stimulating cell signaling covalent conjugate with doxorubicin through acid labile linker chemotherapeutic nucleoside analogue gemcitabine (dFdC) Au NPs

[80, 81]

[29, 68, 69] in vitro and in vivo therapy (xenograft) [70] in vitro therapy [71]

[72]

in vitro and in vivo therapy (xenograft and prostate tissue) in vitro therapy

[73] [74]

in vitro therapy in vitro and in vivo therapy (xenograft)

[75] [31]

in vitro therapy in vitro therapy

[76]

in vitro therapy

[77]

in vitro therapy

[78] [79]

in vitro therapy proof of principle study in vitro therapy and imaging in vitro therapy

[82] [83] [84]

proof of principle study in vitro therapy and imaging

[85]

in vitro therapy

[86]

in vitro therapy

[87]

in vitro therapy

[88]

in vitro therapy

[89]

proof of principle study in vitro and in vivo therapy (humanized mice) in vitro therapy

siRNA chimera

[90]

siRNA–pRNA nanostructure

[91, 92]

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Table 1. (Continued) Target

Aptamers

Type

SELEX Method

nucleolin

AS1411

DNA

derived from cell growth studies doxorubicin loaded triblock co- [93] implementing G-rich ONTs polymer NPs photosensitizer (TMPyP4) physi- [94] cal conjugate mesoporous silica NPs [95]

Cargo Conjugate

Ref.

cobalt–ferrite NP equipped with [96] radionuclide 67Ga liposomes loaded with anti[97] BRAF siRNAs [98] radionuclide 64Cu

immunoglobulin TDO5 heavy mu chain receptor (IGHMR)

transferrin receptor c2

mucin-1 (MUC1)

DNA

cell-SELEX

RNA, 2’-F-Py in vitro SELEX and cell-SELEX

FB4, GS24

RNA, DNA

GS24 MA3 MUC1-5TR1, MUC1-5TRG2, MUC1-GalNAc3 MUC1-5TR1, MUC1-S1.3/S2.2 MUC1-5TRG2

DNA DNA

in vitro SELEX (nitrocellulose filter retention and affinity spin columns) in vitro SELEX

MUC1-S1.3/S2.2 not declared not declared epidermal growth factor receptor 2 (HER2)

HIV-1 gp120

different aptam- RNA, 2’-F-Py ers named A1, B1, C1, D1 E1 unnamed RNA, 2’-F-Py, 3’-invT HB5 DNA unnamed RNA, 2’-F-Py A-1

cell internalization SELEX

commercial selection, no details

in vitro SELEX in vitro SELEX (immobilized target protein) RNA, 2’-F-Py in vitro SELEX (nitrocellulose filter retention)

PLGA–lecithin–PEG NPs encapsulating paclitaxel or fluorophores dual-functionalized TGN peptide–NPs for glioma-targeted therapy miRNA-221 carrying molecular beacon NPs DNA nanostructures

[99]

doxorubicin encapsulated in mesoporous silica NPs with release trigger by miR-21 micelles of lipid-functionalized aptamer molecules

TTA1

RNA, 2’-F-Py, in vitro SELEX (immobilized 2’-OMe-Pu, target protein) 3’-invT[a]

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in vitro therapy in vitro therapy proof of principle study in vitro and in vivo imaging in vitro and in vivo therapy (xenograft) in vitro and in vivo therapy (xenograft) in vitro therapy and imaging

[100]

in vitro and in vivo therapy and imaging

[101]

[103]

in vitro and in vivo therapy and imaging proof of principle study in vitro therapy

[104]

in vitro imaging

supramolecular reticular DNA– CdTe QDs siRNA-loaded liposomes

[105]

in vitro imaging

[106]

streptavidin-conjugates of proteins targeted to the lysosome

[107]

in vitro therapy and imaging proof of principle study

QD conjugates doxorubicin intercalate photosensitizer (chlorin e6)

[108] [109] [110]

in vitro imaging in vitro therapy in vitro therapy

complement component C1q

[111, 112]

in vitro therapy

icosahedral DNA origami with intercalated doxorubicin radionuclide 99mTc paclitaxel-loaded PLGA NPs miRNA–29b chimera

[113]

in vitro therapy

[114] [115] [116, 117]

in vitro imaging in vitro therapy in vitro and in vivo therapy (xenograft) in vitro therapy

[102]

chimera of miRNA let-7i down- [118] regulating cyclin D1 and D2 chimera of anti-Bcl-2–siRNA [119] with sensitizing effect to cisplatin [120] radionuclide 99mTc intercalated doxorubicin anti-tat/rev siRNA-chimeras

[121] [122] [123] [124]

tenascin-C (TN-C)

Application

radionuclide

99m

Tc

[125]

in vitro therapy

in vitro imaging in vitro therapy in vitro HIV suppression in vitro HIV-suppression in vivo HIV suppression (humanized mice) in vitro and in vivo imaging

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Table 1. (Continued) Target

Aptamers

human interleukin-6 AIR-3A receptor (hIL-6R)

Type

SELEX Method

Cargo Conjugate

Ref.

Application

RNA

in vitro SELEX (immobilized target protein)

photosensitizer (chlorin e6)

[126]

in vitro therapy

streptavidin-conjugates

[127]

hollow Au nanospheres loaded with doxorubicin

[128]

proof of principle study in vitro therapy

human receptor ac- Apt1 tivator of NF-kB (RANK); CD30

RNA, 2’-F-Py, in vitro SELEX (immobilized 2’-OMe-Pu target protein)

avb integrin

RNA, 2’-F-Py in vitro SELEX (immobilized target protein) DNA[b] not declared

Apt-avb3 Apt-avb3

siRNAs encapsulated in PEI NPs [129] chimera with anti-EEF2 siRNA [130]

in vitro therapy in vitro therapy

magnetic NPs for MRI

in vivo imaging (xenograft)

[131]

[a] Internal hexa(ethylene glycol) linker. [b] 2’-Deoxythymidine replaced by 5-N-(benzylcarboxyamide)-2’-deoxyuridine.

endocytosis, macropinocytosis, and phagocytosis). Discrimination between these pathways is possible by exploring their dependencies on certain cellular proteins or lipids, such as kinases, small G proteins, actin, or dynamin.[132] Thus, the uptake mechanism can be identified by inhibiting regulating units that are essential for a given pathway.[133] An example of the analysis of uptake mechanisms is given for the nucleolin binding aptamer AS1411.[134] Another approach to investigate the uptake of a molecule of interest is to co-localize it with markers that are exclusively or mainly internalized by a certain pathway (e.g., transferrin and its receptor for CME).[135] However, it should be noted that mammalian cells contain multiple pathways and that most endocytic cargos enter the cell by more than one of them.[132] There are several brilliant reviews that provide in-depth information on endocytic pathways.[132, 136–138] The majority of surface proteins are actively internalized by receptor-mediated endocytosis (RME), a process that is promoted by clathrin-coated pits in the case of various aptamertargeted proteins including PSMA,[139] epidermal growth factor receptor (EGFR),[140, 141] and transferrin receptor (TfR).[142] The pathway of those receptors proceeds from the mild acidic early endosome (EE; pH 5.9–6.0) where sorting leads to direct recycling back to the cell surface (t1/2 ~ 2.5 min), or maturation of the endocytic vesicle to the late endosome (LE) and lysosome, where further degradation processes occur. Acid-labile linkages such as hydrazones, imines, and acetals promote the efficacy of the conjugate as the drug is segregated from the aptamer–target complex in the EE, preventing its exclusion or degradation. Subsequent endosomal escape will occur for agents that are membrane permeable under mild acidic conditions, such as doxorubicin or chlorin e6. For small interfering RNAs (siRNAs) and other membrane-impermeable therapeutics endosomal escape can be enhanced by means of additives (endosomal escape agents) such as proton sponges,[143, 144] membrane-active proteins[145] or peptides,[146] and photochemical internalization compounds.[147] Alternative conjugation strategies that promote controlled release can be realized by linkages of redox-active groups such as disulfides[75] or bonds susceptible to cleavage by enzymes  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

present in the cytoplasm such as esterases, as has been applied for nanoparticles.[148] siRNAs and other oligonucleotide therapeutics One of the major difficulties for a clinical application of ONT therapeutics such as siRNAs, microRNAs (miRNAs), and antisense ONTs relies on systemic and cell-specific delivery. Given their polyanionic character, nucleic acids are cell-impenetrant, and various adjuvants such as lipids or liposomes,[149, 150] cationic nanoparticles,[151, 152] antibodies,[153–155] and cholesterol conjugates[156, 157] have been used to facilitate transition across cellular membranes. Readers are referred to an excellent review about systemic administration of RNA-based therapeutics by Burnett and Rossi.[158] ONT therapeutics display one reasonable class of drug payload for specific aptamer delivery, as linkage to the aptamer is straightforward and can be achieved by extension of an RNA aptamer and/or annealing of one or more RNA oligos, circumventing further conjugation or purification steps. Those constructs are often referred to as aptamer–siRNA chimeras (AsiCs), as they represent the combination of two noncoding nucleic acids with different functions. In 2006 two research groups independently reported such constructs, both using 2’-F-Py anti-PSMA RNA aptamers. Ellington and co-workers[73] constructed a noncovalent conjugate of two biotinylated A9 anti-PSMA aptamers and two biotinylated dicer substrate siRNAs (DsiRNAs) against lamin A/C or GAPDH connected by a streptavidin unit through biotin–streptavidin interaction (Figure 2 a). After exposure to this conjugate, selective uptake and RNAi activity was observed in PSMA-positive LNCaP cells (with PSMA-negative PC-3 cells as a control). A different conjugation strategy was used by the Sullenger group[68] to deliver siRNAs silencing two genes overexpressed in human tumors, the polo-like kinase 1 (plk1) and bcl-2. The passenger strand of these siRNAs was conjugated to the A10 anti-PSMA aptamer as an extension of the 3’ terminus to which the siRNA guide strand was annealed (Figure 2 b). Upon internalization, these AsiCs induced RNAi-mediated cell death ChemMedChem 0000, 00, 1 – 15

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Figure 2. Selection of reported aptamer–siRNA/shRNA conjugates. a) The Ellington research group designed a noncovalent streptavidin conjugate of two biotinylated anti-PSMA aptamer molecules and two biotinylated 27-mer DsiRNAs.[73] b), c) Effective AsiCs containing the truncated anti-PSMA aptamer A10-3.2 were reported by Dassie et al.[29] The hybrid construct (b) represents an advancement of the first-generation AsiC with the non-truncated aptamer A10.[68] The efficacy was enhanced by swapping siRNA strands and adding a 3’-UU overhang. The stem–loop construct (c) yielded similar RNAi effects. d) Aptamer–shRNA chimeras were reported for aptamers targeting PSMA,[71] avb3 integrin,[130] and MUC1,[116] resembling stem–loop structures of endogenous miRNAs. Wullner et al.[70] designed a similar bivalent construct containing a second aptamer unit at the shRNA stem–loop position. e) Aptamer–stick–siRNA conjugates consist of three strands annealed to one another. This design was first reported by Zhou, Swiderski, and co-workers[123] and was refined by the introduction of carbon linkers to decrease steric hindrance of the DsiRNA processing.[159]

leading to decreased tumor growth and tumor regression in a xenograft model of prostate cancer in mice. Since then, numerous research groups have reported AsiCs of various aptamers and siRNA targets (see Table 1 and Figure 2). The compromised siRNA activity of the aptamer conjugates relative to the free siRNA is a crucial impediment. Therefore, several variations of the A10–PLK1 chimeras were tested systematically by Giangrande et al.[29] Following a rational aptamer truncation (A10-3.2) 2-nt 3’ overhangs (UU) were applied to increase dicer enzyme recognition. Next, a wobble base pair was integrated at the 3’ terminus of the passenger strand to favor the loading of the guide strand into the RNA-induced silencing complex (RISC). Finally, it was found that swapping the siRNA strands within the chimera or designing the chimera as a stem-loop-forming single strand (Figure 2 c), yielded the most pronounced RNAi effect.[29] Furthermore, PEGylation at the 5’ end was found to increase the circulating half-life from < 35 min to > 30 h. The anti-PSMA aptamer A10 and its truncations have been further combined with several other siRNAs by various research groups.[160] For example, Wullner et al. demonstrated bivalent chimeras with a small hairpin RNA (shRNA) (Figure 2 d) against eukaryotic elongation factor 2 (EEF2) to exhibit up to fourfold greater cellular uptake than a monovalent construct.[70] A general efficacy analysis of siRNA chimeras was reported by Berezhnoy et al. in 2012,[161] revealing that the thermal stability of the siRNA is also an important parameter of siRNA activity in its conjugated form. When conjugated to the 3’ end of  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org 2’-F-Py RNA-aptamers, siRNAs with lower melting temperature (Tm) were less affected in their silencing ability. Moreover, the configuration of the aptamer–siRNA conjugate retained activity similar to that of the free siRNA duplex when the passenger strand was co-transcribed with the aptamer and 3’ overhangs on the passenger strand were removed. miRNAs represent another class of ONT therapeutics. The restoration of miRNA levels by specific delivery has attracted attention, as a significant portion of the human genome is regulated by miRNAs, and many reports demonstrate that miRNA expression is deregulated in human cancers.[162] Esposito et al. reported a multifunctional aptamer–miRNA construct for myeloid leukemia therapy.[163] This construct (GL21.T–let) comprised RNA aptamer GL21.T and tumor-suppressing miRNA let7g.[164, 165] GL21.T was previously shown to bind to oncogenic receptor tyrosine kinase Axl (Kd = 12 nm) and to be able to block Axl-dependent tranducing events, including Erk and Akt phosphorylation.[166] Treatment of Axl-positive cells with GL21.T–let resulted in pronounced inhibition of cancer cell survival and migration in cell culture as well as tumor growth inhibition in a xenograft mouse model of human lung cancer. Further deliveries of siRNAs and miRNAs are listed in Table 1 and are discussed in the Nanomaterials section below.

Doxorubicin Doxorubicin (DOX) and its derivatives daunorubicin and epirubicin are anthracycline-based chemotherapeutics that block replication and transcription of genomic DNA by intercalation into double-stranded nucleic acids, preferentially GC or CG sequences (Figure 3). This ability can be used to generate physical conjugates with cell-targeting aptamers, such as anti-PSMA aptamer A10,[63] anti-PTK7 aptamer sgc8,[82] or anti-MUC1 aptamers,[109] the cellular uptake and drug release of which can be monitored by the fluorescence properties of DOX. The membrane permeability of DOX facilitates its sub-localization into the nucleus after uptake and intracellular degradation of the aptamer, but it also causes side effects to non-targeted cells if the drug is detached from the aptamer prior to internalization. An advancement to overcome this difficulty was reported by Boyacioglu et al.,[76] who applied pH-sensitive covalent linkages between intercalated DOX within a dimer of anti-PSMA DNA aptamer SZTI01. Thereby, the half-life for DOX bound to the

Figure 3. Structures of doxorubicin and its derivatives.

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CHEMMEDCHEM REVIEWS aptamer was increased from  5 min to > 8 h under physiological conditions, and controlled release within PSMA-positive cells and nuclear accumulation could be confirmed, enhancing the specificity of the drug conjugate. Other covalent conjugations can be accomplished either directly in the solid-phase synthesis of the aptamer or post-synthetically by thiol- or amino-modified aptamers through cleavable groups such as hydrazones formed by the keto group of DOX.[87, 93, 167]

Photosensitizers Photodynamic therapy (PDT) is a clinically established noninvasive method that destroys target cells by the combination of light and stimulable agents, termed photosensitizers (PS).[168] These compounds can generate singlet oxygen, resulting in reactive oxygen species (ROS) by photoconversion or direct radical formation. Most PS are porphyrins that can be activated by long-wavelength light (600–700 nm), which is suitable for in vivo application, as it prevents side effects caused by photoreactions of other tissue molecules and ensures tissue penetration of the light. PS can be attached to an aptamer by covalent linkage using EDC/NHS chemistry. Chlorin e6 has been used for such conjugates with various aptamers, as it offers three carboxylic groups for an amide linkage to a terminally amino-modified nucleic acid (Figure 4).[110, 126, 169, 170] An alternative is the construction of a physical conjugate between the aptamer and the PS. Cationic porphyrins, such as TMPyP4 (Figure 4), were found to bind to nucleic acids, in particular to G-quadruplexes, by stacking with the G-tetrads.[171] Thus, aptamers with G-quadruplex structures can serve as carriers for these PS. The nucleolin binding DNA aptamer AS1411 was used by Shieh et al. to deliver TMPyP4 into MCF-7 breast cancer cells and induce specific photodamage upon irradiation.[94] A promising combination of PDT and photothermal therapy was reported by Wang et al.[78, 172] using multimodal constructs on the basis of gold nanorods. The leukemia T-cell-specific aptamer sgc8 was conjugated by thiol–Au covalent bonds onto the nanorod surface, acting as a recognition tool as well

Figure 4. Structures of photosensitizers chlorin e6 and TMPyP4.

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www.chemmedchem.org as a switch to turn on chlorin e6 by moving it away from the nanorod in the presence of the aptamer target. Nanomaterials Nanomaterials, including micelles, liposomes, dendrimers, and nanotubes or nanoparticles (NP) built from inorganic or organic polymers, lipids, proteins, or nucleic acids are in use for many applications of enhanced drug delivery to tumors.[173] Functionalization with aptamers improves the specificity of nanoparticles and has been reported in several studies for target cell imaging,[174] active targeted therapy[89, 175] or highly efficient cell sorting.[176] NPs can also function as cargo carriers for various kinds of encapsulated therapeutics.[177] Such approaches bear the advantage of multiplying the effective drug amount per aptamer molecule and creating higher target affinities by multivalency effects.[80, 83, 178] Therefore, NP platforms have been exploited extensively in recent years and represent promising tools for future therapeutic applications. The first proof-of-principle study of a delivery through aptamer–NPs was reported by Farokhzad et al. in 2004[179] using the anti-PSMA aptamer A10 conjugated to poly(lactic acid)–PEG copolymer nanoparticles. In combination with aptamer A10, several chemotherapeutics were tested as drug cargos; for example, the cytotoxic drugs docetaxel and paclitaxel were embedded in biodegradable poly(d,l-lactic-co-glycolic acid)-block-poly(ethylene glycol) polymers[57–59] or PtIV-containing precursors of cisplatin, which were shown to be converted into cytotoxic PtII species by the reducing environment within the cytoplasm.[61] A majority of studies addressed the delivery of DOX, for example, in combination with quantum dots (QDs)[167] or with immune-stimulating dinucleotide CpG on polyamidoamine (PAMAM) dendrimers (Figure 5 a).[74] Two examples of so-called DNA origami, wire works of complementary DNA strands, were reported for the transport of multiple DOX units into cancer cells. Chang et al.[113] constructed a DOX-intercalated icosahedral DNA nanoparticle conjugated with MUC1 binding DNA aptamers and observed specific internalization and toxicity of the construct in MUC1-positive MCF-7 cells (Figure 5 b). Zhu et al. used DNA nanostrains, tandem dsDNA built from repetitions of short overlapping single strands, tethered with PTK7 binding DNA aptamer sgc8 to deliver the intercalated drug into CEM cells and induce cell death (Figure 5 c).[77] In a similar fashion, hollow gold nanospheres (HAuNS) served as carriers for DOX. These 42-nm particles were functionalized with CD30 binding RNA aptamers to tackle different CD30-expressing lymphoma cells (Figure 5 d).[128] HAuNS were equipped with DOX by electrostatic interactions of the cationic amino residue to negatively charged citrates on the nanoparticle.[180] In all three studies, a linkage susceptible to decreased pH in the endosome/lysosome was chosen to promote intracellular release of the drug. If the intracellular pathway of the target itself already conveys the conjugate into the cytosol (e.g., nucleolin), further controlled release strategies can enhance the efficacy and specificity of the therapeutic. An interesting trigger for drug reChemMedChem 0000, 00, 1 – 15

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Figure 5. Selection of nanomaterial conjugates. a) Dendrimeric polyamidoamine (PAMAM) has been used for chemoimmunotherapeutic co-delivery. CpG-containing oligonucleotides (CpG ONTs) were covalently linked to the PAMAM dendrimers. The anti-PSMA aptamer A9 was annealed to ONTs, and doxorubicin intercalated into the resulting double-strand regions.[74] b) Schematic of DNA icosahedra with implemented MUC1 aptamer acting as cellspecific carriers for intercalated doxorubicin.[113] c) Aptamer-tethered DNA nanostrains constructed from an ONT containing anti-PTK7 aptamer sgc8 and two building block ONTs annealing as repetitive “boxcars” to carry multiple doxorubicin units.[77] d) Schematic of hollow gold nanospheres equipped with anti-CD30 RNA aptamer. The particles were used as carriers for doxorubicin that was loaded via electrostatic interactions to the citratecoated surface.[128] e) Schematic of a nanocarrier consisting of capped mesoporous silica-coated quantum dots loaded with doxorubicin. The capping was achieved by DNA hybrids of ONTs comprising the sequence of anti-nucleolin aptamer AS1411 and anti-miR-21 ONTs, mediating cell-specific targeting and intracellular drug release.[50] f) Rationally designed nanocarriers. Quantum dots were noncovalently coated with cationic polyethyleneimine (PEI) and loaded with thiol-modified siRNAs by electrostatic interactions. Anti-PSMA aptamer was finally introduced to the siRNA by disulfide linking.[31] g) Schematic of nanocarriers for co-delivery of siRNAs/shRNAs and intercalated doxorubicin. Anti-PSMA aptamers were covalently linked to branched polyethyleneimine–polyethylene glycol (PEI–PEG) co-polymers.[67]

lease in the cytoplasm was used by Zhang et al.[103] using an overexpressed oncogenic miRNA (miR-21) as an intracellular trigger for the release of DOX from mesoporous silica NPs that were gated into cancer cells by the nucleolin binding aptamer AS1411. The pores of the drug-loaded particles were sealed with anchor DNA of anti-miR-21 oligos. As a consequence, drug release was activated by competitive annealing of miR-21 present in the cytosol (Figure 5 e).  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org One of the most sophisticated strategies using nanoarchitecture was reported by Douglas et al.[86] The authors developed a DNA-origami-based nanorobot, a hexagonal capsule consisting of two hinged half-barrels of 35  35  45 nm size, that could be opened in the presence of a certain aptamer target. The corresponding aptamer served as a lock, promoting exclusive release of the payload stowed within the capsule to cells exposing the target receptor. Finally, aptamer-modified nanocarriers have come into focus for the delivery of siRNAs, shRNAs, and miRNAs.[181] In addition to the aforementioned benefits, solutions can be provided for drug release from the endosome, as it is particularly necessary for therapeutic nucleic acids. Bagalkot and Gao[31] reported rationally designed poly(maleic anhydride-alt-1-tetradecene) (PMAT)-coated CdSe/ZnS core shell QDs as a delivery platform for AsiCs in PSMA-positive C4-2B cells (a lineage-derived LNCaP subline). First the QDs were covered with positively charged polyethyleneimine (PEI) and subsequently decorated with antiEGFP siRNAs by electrostatic interactions, thereby reducing the positive charges on the nanoparticle surface. Subsequently, the aptamer A10 was linked via a cleavable disulfide bridge to the siRNA. This ensured proper folding of the aptamer and also provided the intracellular secession of the aptamer. Acidic pH within the endosome triggered siRNA detachment from the nanoparticle surface, yielding cationic PEI-covered QDs that could disrupt the endosomal vesicle to release the siRNA into the cytosol (Figure 5 f). The construction of self-assembling multifunctional RNA nanomaterials was established by Guo et al. using packing RNAs (pRNAs), deduced from bacteriophage phi29 small RNAs, in combination with anti-CD4 aptamers and siRNAs.[91, 92, 182] The applied phi29 pRNA domain contains two loops that are responsible for hand-in-hand interaction between each other through intermolecular base pairing,[183, 184] allowing its use as a building block for dimers, trimers, hexamers, or higher-ordered nanostructures. The 5’-/3’-helical domain of phi29 pRNA can be extended by RNA aptamers, siRNAs, or ribozymes without loss of the packing ability to build multifunctional therapeutic nanocarriers. The idea of harnessing nanocarriers for combinatorial drug delivery has also been followed in other studies. Kim et al.[67] constructed polyplexes consisting of branched PEI grafted to polyethylene glycol (PEG) for co-delivery of DOX and a shRNA against Bcl-xL into PSMA-positive cells leading to synergistic effects that induced apoptosis. 5’-Amino-modified anti-PSMA aptamer A10 was conjugated by a sulfo-SMCC linker to the thiol-containing polymer strains, and DOX was intercalated into the helical part of the aptamer RNA afterward. These conjugates could be complexed with the shRNA by electrostatic interactions to yield the final cell-specific and endosome-disrupting polyplexes (Figure 5 g). Further miscellaneous therapeutics Ribosome-inactivating proteins (RIP), mostly from plants, are a class of toxins with glycosylase activity; their cytotoxicity rests on depurination of ribosomal RNA.[185] They are divided ChemMedChem 0000, 00, 1 – 15

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CHEMMEDCHEM REVIEWS into two major classes regarding their ability to actively enter cells. Type 1 RIPs, such as gelonin or saporin, only comprise the enzymatically active A chain (30 kDa) and have only little toxicity relative to type 2 RIPs, such as ricin or abrin, that exhibit an additional B chain (35 kDa) with lectin properties to specifically bind to glycans on the cell surface. Gelonin has been applied in aptamer-directed delivery by Ellington and colleagues as a conjugate with amino-modified anti-PSMA aptamer A9.[75] The aptamer was bridged to a reduced cysteine residue of the toxin through a redox-active disulfide bond of a SPDP cross-linker. The resulting aptamer–toxin conjugate had an IC50 value of 27 nm and displayed increased potency by at least 600-fold relative to PSMA-negative cells. Radionuclides have also been reported as an aptamer cargo for active tumor targeting. The 39-nt RNA aptamer TTA1 was selected against purified tenascin-C (TN-C), an extracellular matrix protein overexpressed in the tumor stroma and involved in oncogenesis.[186] TTA1 is also an interesting example of post-SELEX modification to improve in vivo stability. After truncation of the selected 2’-F-Py-RNA aptamer the purines were replaced by 2’-OMe-purines (2’-OMe-Pu) as far as possible. Then an inverted thymidine 3’ cap was added, and an internal section of 17 nt was replaced by a hexa(ethylene glycol) linker. By conjugation of metal-chelating mercaptoacetyldiglycine (MAG2) to the 5’ terminus, the radiolable 99mTc could be complexed with the aptamer. After intravenous injection into glioblastoma (U251)-bearing mice and breast cancer (MDAMB435) tumor xenografts, the rapid uptake of the aptamer enabled clear tumor imaging.[125] Other aptamers have been applied in radionuclide-aided tumor imaging, such as anti-MUC1 aptamer MUC1-S1.3/S2.2,[114] anti-epidermal growth factor receptor 2 (HER2) aptamer,[120] and the anti-nucleolin aptamer AS1411.[98] The latter has also been used for multimodal in vivo tumor imaging using cobalt–ferrite nanoparticles functionalized with chelating agents to carry 67Ga radionuclides. This construct allowed tracking of C6 tumor cells in mice by magnetic resonance imaging (MRI) of the nanoparticles as well as scintigraphic imaging of the radionuclide up to 24 h after injection.[96] Some therapeutic nucleoside analogues can be incorporated into ONTs replacing one of the canonical nucleosides, for example, the cytidine analogue gemcitabine (2’-deoxy-2’,2’-difluorocytidine, dFdC, Gemzar). Gemcitabine is a potent chemotherapeutic, mainly applied to pancreatic cancer therapy, acting as an inhibitor of DNA polymerase in its triphosphorylated form (dFdCTP) and to ribonucleoside reductase in its diphosphorylated form (dFdCDP).[187] The incorporation of gemcitabine into ONTs can be achieved by in vitro transcription with T7 RNA polymerase variant Y639F. Ray et al.[88] used 2’-F-PyRNA aptamer E07, targeting epidermal growth factor receptor (EGFR), to deliver a gemcitabine-containing ONT annealed to the aptamer strand into pancreatic cancer cells (MiaPaCa-2) resulting in a cell-specific decline in proliferation.

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www.chemmedchem.org Summary and Outlook In summary, RNA and DNA aptamers present valuable tools for targeted therapy. The diverse applications reported in cancer therapy using aptamer therapeutic conjugates show great promise for future developments in clinical treatment. So far, pegaptanib (Macugen, Pfizer), a 28-nt 2’-F-Py and 2’-OMe-Pu RNA aptamer targeting vascular endothelial growth factor (VEGF) for the treatment of neovascular age-related macular degeneration, is the only example that has made its way into clinical application.[188] The difficulties of finding aptamers that are specifically and substantially internalized in vivo can be overcome by means of novel cell-based selection approaches. Progress in the SELEX process, such as the use of microfluidic systems, cell-SELEX or assay-based selection, are now enabling researchers to better implement the conditions required for later exertions. Cell internalization SELEX and in vivo SELEX in particular are promising tools for the direct selection of aptamers that are internalized by target cells or malignant target sites, respectively. While SELEX used to be a “black box” with little insight into pool composition, diversity, and enrichment patterns, nextgeneration sequencing allows the whole SELEX to be covered, and even rare aptamers to be identified very early during selection. The generation of aptamers with clinical applicability may also benefit from the fact that many patents associated with aptamers and SELEX are currently expiring. This should simplify the transition from basic laboratory research to commercialization for research institutions and small companies.[189] Having the preparation techniques of nanotechnology in hand, a broad spectrum of drug delivery, most of all rationally inspired co-delivery of synergistic agents such as siRNAs and chemotherapeutics, is currently possible. Nanotechnology also provides solutions for controlled release, the triggered activation or sub-localization inside targeted cells. However, understanding of internalization and trafficking routes of many cellsurface proteins is often incomplete. This can impair prediction of optimal subcellular localization for aptamer–drug conjugates. Advancements regarding these aspects will further improve the efficacy of aptamer-mediated targeted therapy.

Acknowledgements We thank Anne Al-Housami for carefully reading the manuscript. Cover picture: MUC1 aptamer[190] conjugated with chlorin e6 approaching cancer cells.[191] Keywords: antitumor agents · aptamers · conjugation · drug delivery · targeted therapy [1] A. D. Ellington, J. W. Szostak, Nature 1990, 346, 818 – 822. [2] C. Tuerk, L. Gold, Science 1990, 249, 505 – 510. [3] E. J. Cho, J. W. Lee, A. D. Ellington, Annu. Rev. Anal. Chem. 2009, 2, 241 – 264. [4] D. H. Bunka, P. G. Stockley, Nat. Rev. Microbiol. 2006, 4, 588 – 596. [5] E. Levy-Nissenbaum, A. F. Radovic-Moreno, A. Z. Wang, R. Langer, O. C. Farokhzad, Trends Biotechnol. 2008, 26, 442 – 449.

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Received: April 30, 2014 Revised: July 2, 2014 Published online on && &&, 0000

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REVIEWS Selective delivery: Active drug targeting enhances the efficacy and specificity of systemic therapeutics. Aptamers, artificial nucleic acid ligands, represent powerful targeting tools that can act as cell-specific drug carriers. The advancements from the past decade have provided various approaches that open new gateways for drug administration in cancer therapy.

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

S. Kruspe, F. Mittelberger, K. Szameit, U. Hahn* && – && Aptamers as Drug Delivery Vehicles

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