Review

Cell-specific aptamers and their conjugation with nanomaterials for targeted drug delivery 1.

Introduction

2.

Aptamers as molecular recognition probes

3.

SELEX: the in vitro selection process

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4.

Cell-SELEX: cell-based selection of aptamers

5.

Aptamers for biomarker discovery

6.

Aptamers for biomedical applications

7.

Conclusion

8.

Expert opinion

Jie Liao, Bo Liu, Jun Liu, Jiani Zhang, Ke Chen & Huixia Liu† †

Central South University, Xiang ya Hospital, Changsha, Hunan, China

Introduction: Aptamers are short, single-stranded DNA or RNA sequences that can fold into complex secondary and tertiary structures and bind to various target molecules with high affinity and specificity. These properties, as well as rapid tissue penetration and ease of chemical modification, make aptamers ideal recognition elements for in vivo targeted drug delivery and attractive molecules for use in disease diagnosis and therapy. Areas covered: The general properties of aptamers as well as advantages over their counterpart antibodies are briefly discussed. Next, aptamer selection by cell- systematic evolution of ligands by exponential enrichment is described in detail. Finally, the review summarizes recent progress in the field of targeted drug delivery based on aptamers and their conjugation to liposomes, micelles and other nanomaterials. Expert opinion: Advances in nanotechnology have led to new and improved nanomaterials for biomedical applications. Conjugation of nanoparticles (NPs) with aptamers exploits both technologies, making aptamer-NP conjugates ideal agents for drug delivery with proven therapeutic effects and the reduction of toxicity to normal tissue. The use of multivalent aptamerconjugated nanomaterials represents one of the new directions for drug development in the future; as such, continuing studies of these multivalent aptamers and bioconjugates should result in important clinical applications in targeted drug delivery. Keywords: aptamer, cell-systematic evolution of ligands by exponential enrichment, nanomaterials, targeted drug delivery Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

Advances in molecular medicine over the past decades have helped to improve our understanding of the complex interplay among genetic, transcriptional and translational alterations in human diseases [1]. These molecular changes form the basis for an evolving field of targeted therapy and diagnosis. In the current era of molecular medicine, molecular probes are indispensable as signaling molecules for the localization and quantification of structural and functional components in disease processes. These probes can be used to interrogate the molecular basis of disease pathogenesis, facilitate disease diagnosis, define the extent of disease, implement a tailored therapeutic regimen and even monitor treatment response. Proteins, mainly antibodies, are the most studied molecular probes. Although mAbs have been widely used as escort molecules for the targeted delivery of drugs and nanomaterials, several limitations, including large size, high manufacturing cost, batch-to-batch variations, difficult conjugation and high immunogenicity have hampered their use. Other molecular probe candidates include those from peptides and small molecules, some of which have been clinically used for disease diagnosis and therapy [2]. However, an emerging group of molecules that holds 10.1517/17425247.2015.966681 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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J. Liao et al.

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Aptamers are single-stranded DNAs or RNAs that are able to recognize targets with high affinity and specificity, making them ideal recognition elements for targeted therapy. Cell-systematic evolution of ligands by exponential enrichment (cell-SELEX), whereby live cells are used to develop an aptamer that will bind specifically to a particular target cell without prior knowledge of molecular signatures on the cell surface, provides an effective approach for the discovery of new biomarkers of cancer cells. Aptamers can be directly used to modulate the biological activities of their targets or can be easily modified for chemical conjugation with a host of functional moieties, including drug-carrying nanoparticles (NPs), micelles, nanotrains and siRNA. This allows specific recognition of the target cancer cells and reducing damage to nearby normal cells at the same time, showing high potential for drug delivery in cancer therapy. Different strategies of conjugation of NPs with aptamers for the targeted therapy are discussed. Conjugation of nanomaterials with aptamers for targeted therapy has the potential to improve the therapeutic outcome of chemotherapy.

This box summarizes key points contained in the article.

Aptamer

Protein

modification, aptamers have attracted considerable attention in the field of probe development [3]. Recently, a simple, fast and reproducible cell-based aptamer selection strategy called cell-systematic evolution of ligands by exponential enrichment (cell-SELEX), using whole, intact cells as the target for aptamer selection has been developed. This selection process then generates multiple aptamers for the specific recognition of biological cells, but without the need for prior knowledge about the signature of target cell-surface molecules. Thus, aptamers selected via cell-SELEX, or proteinSELEX, have superior potential as carriers for the targeted delivery of drugs. This review focuses on the development of receptor-specific aptamers and their application in targeted drug delivery, as well as their potential benefits and drawbacks. 2.

In 1990, Gold and Szostak [4,5] independently reported the selection of aptamers identified from an initial library containing 1013 -- 1016 random ssRNA or ssDNA sequences through an in vitro selection process called SELEX. Since then, aptamers have attracted increasing interest in the scientific community, and they have been widely applied in a variety of fields, including biomedical and cellular engineering. Derived from the Latin aptus, the term aptamer means ‘to fit’ [6], implying the key-and-lock relationship of this class of molecules with their targets (Figure 1) [7]. For their highly selective and specific target recognition and binding, they are usually called chemical antibodies. Aptamers are analogous to antibodies in their range of target recognition and variety of applications. However, as molecular probes, they possess a number of superior attributes over antibodies, including: i) chemical integrity at ambient temperature and in extreme pH and organic solvents [8-10]; ii) wider range of target molecules, including metal ions, nucleotides, toxins, enzymes, amino acids, proteins, viruses, bacteria or even whole cells (Table 1) [11-25]; iii) lower immunogenicity; iv) easier and more reproducible production; v) easier chemical modification with electrochemical probes, fluorophores, quenchers, and antibodies or functional groups to yield improved, custom-tailored properties [26-28]; and vi) capability as biosensors because of their conformational variations, such as G-quadruplex. 3.

Figure 1. Three-dimensional structures of aptamer and target protein.

the most promise is a new class of designer nucleic acids (NA), termed aptamers, which are single-stranded DNA/RNA that are able to recognize specific targets, such as single proteins and even small molecules. Owing to their small size, low immunogenicity, and ease of manufacture and chemical 2

Aptamers as molecular recognition probes

SELEX: the in vitro selection process

Typically generated through the SELEX process, > 150 aptamer targets have been identified, such as cocaine, peptides, growth factors, viral proteins and toxins as well as cells and bacteria [29-32]. In general, the SELEX process includes three iterative steps to obtain the best target-binding nucleotides. The first step involves the preparation of a library consisting of at least 1014 -- 1015 random DNA or RNA sequences, usually 30 -- 40 mers, flanked on both sides with fixed primer

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Cell-specific aptamers and their conjugation with nanomaterials for targeted drug delivery

Table 1. Recent examples of aptamers isolated against various targets. Target class

Aptamer types

Small molecules

RNA RNA RNA DNA RNA DNA RNA DNA DNA DNA RNA DNA DNA DNA DNA

Viral

Bacterial

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Adhesion proteins, molecules, receptors Cells

Target name

Ref.

L-arginine Aromatic amines Codeine Thrombin HIV gp 120 GD protein of herpes simplex virus 1 Hepatitis B virus P protein Lactobacillus acidophilus Staphylococcus aureus VEGF Prostate-specific membrane antigen Fibronectin T cell acute lymphoblastic leukemia cell line, CCRF-CEM Ramos cells Ovarian clear cell carcinoma subtype

[11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

sequences for amplification. The second step involves binding and separation, whereby the library is exposed to the target, resulting in target-bound library components, which are then separated from the unbound components. This step is generally coupled with several other methods, including flow cytometry [33], capillary electrophoresis [34], surface plasmon resonance (SPR) [35] and atomic force microscopy [36,37], to increase the efficiency and throughput of aptamer isolation. In the third step, the target-bound library components are polymerase chain reaction (PCR)-amplified to generate a new library for the next round of some 10 -- 20 rounds of selection. In this review, we highlight the cell-SELEX strategy, which selects aptamers able to bind to whole cells and is therefore of particular interest for applications in targeted drug delivery.

Cell-SELEX: cell-based selection of aptamers

4.

Cell-SELEX is the process whereby live cells are used to develop an aptamer that will bind specifically to a particular target cell population [38]. Compared with protein-based SELEX, cell-SELEX is a particularly promising selection strategy because it selects aptamers that preferentially bind to diseased cells, especially cancer cells. Thus, these aptamers are capable of functioning as molecular probes that can recognize protein markers overexpressed on the cancer cell membrane for early diagnosis and targeted intervention. In addition, cell-SELEX can be carried out without prior knowledge of the types of proteins on the cell surface or their molecular signatures, providing an effective approach for the discovery of new biomarkers for cancer cells. A typical cell-SELEX procedure is as follows. First, suspension cells are prepared by using a nonenzymatic dissociation solution for digestion. After centrifugation and washing steps to remove culture media, collected cells are incubated with target cells for positive selection. Second, bound sequences

are collected using heat denaturation after unbound probes are carefully washed away. Third, the enriched pool is amplified by PCR and used for the next round of selection or for enrichment monitoring by flow cytometry. During negative selection, the separated single-strand DNAs are incubated with control cells for a time certain. After centrifugation, unbound sequences are collected, effectively removing sequences that interact with commonly expressed proteins and leaving only those sequences that bind with the target cell for selective enrichment. The enriched sequences are then PCR-amplified and ssDNA is obtained from the PCR product via a sodium hydroxide-based separation method. The enriched ssDNA pool is then used for enrichment monitoring by flow cytometry or for the next round of selection. Generally, 15 -- 25 rounds of selection are needed for successful pool enrichment, which depends on partitioning/ amplification steps. The enriched pool is then sequenced and those high-abundance sequences are subsequently chemically synthesized and labeled with fluorophores for aptamer identification. Over the years, the Tan group has successfully generated panels of aptamer probes from cell-SELEX with high affinity and specificity for several types of cancer cells, including small and non-small cell lung cancer, myeloid leukemia, lymphocytic leukemia and liver cancer [23,24,39-41]. 5.

Aptamers for biomarker discovery

Aptamers selected through cell-SELEX can be useful in discovering disease-specific marker proteins that are differentially expressed on certain types of cells. In contrast to conventional methods, such as phage display antibody production and protein-SELEX, which target a previously known specific protein, the novelty of cell-SELEX-based protein discovery is rooted in finding cell-surface membrane markers without prior knowledge of their molecular signature on the cell surface. Aptamers selected via cell-SELEX feature facile chemical modification, ensuring the binding stability and strength

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J. Liao et al.

required for the effective capture, enrichment, and identification of target proteins on the cell membrane surface. Using the cell-SELEX technology, the Tan group has identified a panel of cell-specific aptamers that can also be used as probes for cell-surface biomarker discovery [41,42]. For example, using T-cell acute lymphoblastic leukemia (T-ALL) as a cell-SELEX target, Tan et al. generated a panel of DNA aptamers that recognized a series of cultured cell lines and clinical patient samples [23]. Through binding tests, it was found that one of the selected aptamers, sgc8, showed high specificity and affinity for most T-ALL cells and acute myeloid leukemia (AML) cells, as well as some B-cell acute lymphoblastic leukemia cells [23]. When conjugated with magnetic beads, the aptamer (sgc8)-conjugated magnetic beads (apt-MBs) were used to capture and purify the binding targets of sgc8 on the leukemia cell surface [42]. Combined with mass spectrometry and protein overexpression experiments, protein tyrosine kinase (PTK7) was ultimately identified as a potential biomarker for T-ALL [42]. As another example, TD05 was selected from Ramos cells, a B cell lymphoma cell line, and Immunoglobulin Heavy mu chain (IGHM) was identified as the aptamer’s cognate target [41]. These results demonstrated that aptamers generated using cell-SELEX can serve as highaffinity and specific probes for the identified biomarkers. Moreover, in comparison to antibody-based techniques, aptamer-based biomarker discovery is less labor-intensive and more cost--effective. 6.

Aptamers for biomedical applications

Aptamers as therapeutics Based on their wide spectrum of cellular targets, interest in developing aptamers for use as therapeutic agents has increased, in particular because of their ability to modulate the biological activities of their pathogenic targets. The appearance of pegaptanib (marketed as MACUGEN) was a major milestone in the use of aptamers in clinical therapy. Pegaptanib, an anti-VEGF 165 (VEGF165) aptamer that can recognize most human VEGFA isoforms, was developed by Pfizer and Eyetech, and on 20 December 2004, it was approved by the US FDA for the treatment of age-related macular degeneration [43]. The first therapeutic application was reported by Sullenger et al. [44] who found that the NA could prevent the activation of viral gene expression by overexpressing a trans-activation response decoy in host cells, resulting in the inhibition of viral replication. Anticoagulants are important in surgery, but heparin, the most commonly used anticoagulant, has been found to cause many complications, including nonspecific plasma binding and platelet aggregations. To address these problems and achieve higher efficacy and safety, Dobrovolsky et al. [45] developed DNA aptamers against thrombin to prevent thrombin-induced clotting, as well as platelet cell aggregation. Moreover, Hasegawa et al. [46] demonstrated dimerization of aptamers against different areas of thrombin, which allowed 6.1

4

the enhanced inhibitory action compared with the single anti-thrombin aptamer. So far, many of the aptamers used as anticoagulants are in the first phase of clinical trials. It has become apparent that certain guanine-rich (G-rich) DNA and RNA molecules can associate intermolecularly or intramolecularly to form ‘quadruplex’ structures that result in unusual biophysical and biological properties, such as enhanced cellular internalization and protein binding via shape-specific recognition [47]. AS1411, a G-rich aptamer, has shown the most robust inhibition of cancer cell proliferation thus far based on its binding of nucleolin, as well as its internalization into target cells to interfere with intracellular pathways, including the destabilization of BCL-2 mRNA and the inhibition of NF-kB [48]. AS1411 is currently in Phase II clinical trials for AML [49]. It represents a novel way to target cancer cells at the molecular level and provides a broad range of diagnostic and therapeutic imaging views in cancer medicine. In sum, aptamers, which are currently under clinical evaluation for their efficacy against cell proliferation, infection, inflammation and coagulation are likely to make a direct and significant contribution to the treatment of infectious diseases and cancer in the near future. Aptamers for targeted delivery of therapeutics Although aptamers have shown promise in transporting small molecules, including drugs and siRNA, through the microvasculature or the tumor interstitium to targeted tumor cells, their relatively small size limits the amount of cargo that each aptamer can deliver. Therefore, strategies have been developed to incorporate aptamers as targeting moieties for a variety of nanoparticles (NPs). NPs provide large surface areas that offer excellent platforms for conjugating multiple aptamers and interior volumes that can be used to store large quantities of drug molecules for targeted drug delivery. NPs typically have at least one dimension < 100 nm in size. Compared with other materials, NPs possess large surfacearea-to-volume ratios, unique optical, magnetic, electronic, mechanical, physical and chemical properties [50-53] as well as the ability to be taken up by cells through endocytosis. These properties enable NPs to penetrate a tumor mass, making them ideal candidates for applications ranging from in vivo imaging and diagnosis to therapeutics, especially drug delivery. Chemotherapy is an important treatment of cancer with one or more cytotoxic antineoplastic drugs that can kill the tumor cells, but chemotherapy suffers from the ‘off-target’ effect, resulting in many side effects and toxicity to normal cells or even treatment failure. However, aptamers, with their specificity, high affinity and lack of immunogenicity can be easily modified for chemical conjugation with a host of functional moieties, including drug-carrying NPs, making it possible to recognize the target cancer cell with high specificity and, at the same time, reducing damage to nearby normal cells, thus showing high potential for drug delivery in cancer therapy. 6.2

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Cell-specific aptamers and their conjugation with nanomaterials for targeted drug delivery

A. Doxorubicin

B.

Acid-labile linker

sgc8c

C.

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Sgc8c–Dox conjugates

Intercalation

+

Aptamer

Dox

Aptamer-dox physical conjugate

Figure 2. (A) The molecular structure of doxorubicin with flat aromatic rings (Dox). (B) Schematic representation of noncovalent intercalation of doxorubicin (Dox) into aptamer dsDNA regions. (C) Schematic representation of doxorubicin (Dox) conjugated with aptamer sgc8 through acid-labile linkage for targeted drug delivery.

6.2.1

Aptamer-drug intercalation with doxorubicin

Doxorubicin (Dox) is an anthracycline-derived drug used to treat a wide range of cancers, but it suffers from such toxic effects as hair loss, nausea, dilated cardiomyopathy or congestive heart failure [54]. The high recognition specificity and selectivity of aptamers make them excellent candidates for bioconjugation with Dox for targeted delivery, resulting in improved therapeutic efficacy and reduced toxic effects to normal tissues. It has been reported that Dox and other closely associated drugs, such as daunorubicin, can form a physical complex with aptamers through noncovalent intercalation into dsDNA regions by the presence of flat aromatic rings in the molecule (Figure 2) [55]. A10, an anti-prostate-specific membrane antigen (PSMA) 2¢-fluoropyrimidine-modified RNA aptamer, was first selected by Lupold and showed specific binding to PSMA [56]. The finding that PSMA is internalized via clathrin-coated pits to endosomes suggested that PSMA-specific aptamers may be potential drug delivery carriers [57]. Bagalkot et al. [58] subsequently developed physical aptamer-Dox conjugates for targeted drug delivery (Figure 2). The result demonstrated that aptamerDox conjugates could be internalized into the PSMA-positive prostate cancer cell line LNCaP accompanied by a significant reduction of cell proliferation, as well as enhanced cytotoxicity, in comparison to control cells (n = 5, p < 0.005). In clinical cases, the presence of different cancer subtypes with different heterogeneous biomarkers makes it impossible for one single aptamer to distinguish all clinical samples from different patients, even those with the same type of cancer. To address heterogeneity among cancer subtypes for

targeted drug delivery, the Tan group developed a drug carrier with a broader recognition range of cancer subtypes [59]. Here, two different aptamers, sgc8c and sgc5a, were self-assembled through a double-stranded linker with preferential Dox loading sites. Results showed the bispecific abilities of these aptamers for binding and drug delivery, as well as the induction of bispecific cytotoxicity in target cells and cell mixtures. Recently, Hu et al. [60] developed a drug delivery system by intercalating Dox into the mucin 1 (MUC1) aptamer. This aptamer-drug complex could carry Dox into MUC1-positive tumor cells and, at the same time, significantly reduce drug intake by MUC1-negative cells, in turn suggesting that the MUC1 aptamer may be a potential targeting ligand for selective delivery of cytotoxic agent to MUC1-expressing tumors (n = 6, p < 0.01). Meng et al. [61] developed a targeted delivery strategy by conjugating Dox with a modified DNA aptamer, termed TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. These conjugates exhibited potency, target specificity, as well as fewer toxic effects to the nontarget cell line. Although this physical interaction requires no modification of the drug or the aptamer, it does pose a risk to the efficacy and safety profile of the drugs and/or the binding characteristics of the aptamer. Aptamer-drug conjugation with DOX In order to improve the selectivity of cancer therapy and reduce the toxic effects to normal tissues, mAbs have traditionally been conjugated with drugs for targeted cancer 6.2.2

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A.

B.

C.

2h

12 h

Aptamer 10 μm

PEG Lipid tail

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Self assembling

50 μm

Biotin

D.

E. Post-labeled with QDstreptavidin

2h

Doped special dye Hydrophilic part of micelle Hydrophobic part of micelle F.

0′

50 μm

50 μm

2′

4′

5 μm

8′

Figure 3. (A) Schematic representation of dye-doped micelles. Fluorescent images of Ramos cells after incubation with free CellTracker Green BODIPY for (B) 2 h and (C) 12 h, or (D) incubation with biotin-TDO5-micelle doped with CellTracker Green BODIPY for 2 h. (E) Enlarged fluorescent image after post-labeling the biotinylated TDO5 aptamer with QD705 streptavidin. The inset in image (E) is the fluorescent image of the dead cell. (F) Real-time monitoring of doped special dyes released from the core of the micelles and activated by intracellular enzymes.

therapy. For example, the humanized anti-CD33 antibodycalicheamicin conjugate Mylotarg has already been approved for the treatment of AML. However, with the emergence of aptamer molecules, the Tan group used cell-SELEX to generate sgc8, an aptamer with high affinity and specificity to human T-lymphoblastoid cell line CCRF-CEM, subsequently identifying the target of sgc8, PTK7, thus rivaling mAbs as target recognition elements. In later studies, it was demonstrated that aptamer sgc8 could be internalized into target cells [62,63], indicating the potential use of sgc8 for drug delivery. Accordingly, Tan et al. [64]. have designed an sgc8--Dox conjugate through acid-labile linkage for targeted drug delivery to CCRF-CEM cells by combining the advantages of specific recognition of aptamer sgc8 for PTK7 and the benefits of Dox (Figure 2). The result shows that the conjugate was able to specifically deliver the drug to target CCRF-CEM cells, but not nontarget cells, with dissociation constants (Kd) as low as 2.0 ± 0.2 nM. Furthermore, the conjugates were cleaved inside the acidic endosomal environment and showed a 6.7-fold increase in toxicity to CCRF-CEM cells compared with nontarget cells, indicating promising application in drug delivery. Boyacioglu et al. [65] developed dimeric aptamer complexes (DACs) for specific delivery of Dox to PSMA+ cancer cells. Dox was covalently bound in DACs using a reversible linker that promotes covalent attachment of Dox to genomic DNA following cell internalization. Results showed that DACDox was selectively cytotoxic to C4 -- 2 cells with cytotoxicity similar to that of the molar equivalent of free Dox, whereas it displayed only minimal cytotoxicity to PC3 cells. 6

Aptamer-micelle conjugates for targeted drug delivery

6.2.3

Micelles are aggregates of surfactant molecules with a hydrophobic core in the interior serving as a reservoir for hydrophobic drugs and a hydrophilic shell facing outward to the surrounding solvent that stabilizes the hydrophobic core and renders the polymers water-soluble [66]. Micelles are widely used in biological fields for their ability to dissolve and move nonpolar substances through an aqueous medium or to carry a variety of cargos to cells, such as antisense oligonucleotides and drug molecules [67]. Using cell-SELEX, the Tan group first selected aptamer TD05, which showed remarkable specificity in binding to Ramos, a Burkitt’s lymphoma cell line. Later, the target that bound to aptamer TD05 was isolated and identified by mass spectrometric analysis to be IGHM. As TD05 was selected at a temperature of 4 C, it was found to lose affinity at 37 C. However, Wu et al. [68] developed an aptamer--micelle to target specific cancer cells with efficient detection/delivery by linking TD05 with a PEG and lipid tail that showed strong binding to Ramos cells, even at 37 C. Afterwards, the aptamer-micelle was loaded with the dye CellTrackerTM Green, which only fluoresces inside the cell. After incubating this dye-loaded aptamer--micelle with Ramos cells, a strong fluorescence inside Ramos cells was observed after only 2 h compared with free CellTrackerTM Green, which required 12 h. Most importantly, the results demonstrated that the aptamer--micelle could target cells in flow channel systems, thereby mimicking drug delivery in a flowing system. These advantages demonstrated that the unique assembly

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Cell-specific aptamers and their conjugation with nanomaterials for targeted drug delivery

A.

Boxcars

Self-assembly No reaction 2

100 nm Short DNA building blocks

3

100 nm

100 nm

Aptamer-tethered DNA nanotrains (AptNTrs)

AptNTrs loaded with molecular drugs (drug fluorescence “OFF”)

Transportation with molecular drugs

B.

Target cell membrane n

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1

Drug loading by intercalation

Boxcars loaded with drugs

Chimeric aptamer-trigger

M2

M1

n

1

3 n

2

Receptor Endocytosis Drug unloading Drug fluorescence “ON”

Nucleus

Figure 4. Schematic representation of the self-assembly of aptamer-tethered DNA nanotrains (aptNTrs). (A) Self-assembly of aptNTrs from short DNA building blocks (1) upon initiation from a chimeric aptamer-tethered trigger probe. The resultant long nanotrains (2) were tethered on one end with aptamers working as locomotives hauling multiple repetitive drug-loaded ‘boxcars’ (3). (B) The drugs were specifically transported to target cancer cells via aptNTrs and subsequently unloaded, inducing cytotoxicity to target cells. The fluorescence of drugs loaded onto nanotrains was quenched (‘OFF’), but it was recovered upon drug unloading (‘ON’), enabling this platform to signal target recognition and drug delivery.

possesses the capacity for rapid molecular recognition of cancer cells and in vivo drug delivery (Figure 3). Farokhzad et al. [69] reported an RNA aptamer--micelle for PSMA to target prostate tumors and it was shown that these NPs specifically bound to PSA-expressing cancer cells, inducing a 77-fold increase versus the control group. They next loaded the aptamer--micelle with docetaxel and examined treatment in prostate cancer [8]. Results showed that the aptamer-encoded micelles had a significantly increased cytotoxicity over nontargeted counterparts in in vitro assays using LNCaP prostate cancer cells. Moreover, in in vivo studies using an LNCaP xenograft nude mouse model, the aptamerencoded micelles showed a significant increase in antitumor

efficacy as well as lesser levels of systemic toxicity over the nontarget control (n = 150, p > 0.001). Aptamer nanotrains for targeted drug delivery Whereas traditional nanomaterials have the potential to deliver chemotherapeutic drugs with higher dosage, such nanomaterials have not produced highly therapeutic effects as a result of their passive delivery methods. However, as discussed above, the emergence of aptamer-nanomaterial bioconjugates has improved therapeutic efficacy and reduced toxicity. As some nanomaterials are expensive and as drug tolerance is always an issue, the Tan group worked on a strategy to attain maximum tolerated doses [59] and developed a 6.2.4

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simple, target-specific, economical and biocompatible drug delivery platform termed aptamer-tethered DNA nanotrains (aptNTrs) for targeted cancer therapy (Figure 4). Upon initiation from a chimeric aptamer-tethered trigger probe, aptNTrs are self-assembled from short DNA building blocks. The resultant long nanotrains are tethered on one end with aptamers working as locomotives hauling multiple repetitive drug-loaded ‘boxcars’. Drugs are specifically transported to target cancer cells via aptNTrs and subsequently unloaded, inducing cytotoxicity to target cells. These aptNTrs demonstrated potent antitumor efficacy and reduced side effects in a mouse xenograft tumor model, making them promising targeted drug transport platforms for cancer theranostics. In addition, the DNA aptamers forming the train’s locomotive are interchangeable; thus, it is possible to recognize and target different types of cancers, largely expanding the application of these aptNTrs. Aptamer-based conjugates for photothermal therapy

6.2.5

Photothermal therapy (PTT), in which photon energy is converted into heat to destroy cancer cells, is a less invasive therapeutic intervention against cancer cell proliferation because of the low absorption efficiency of natural tissue absorbents. In the past, synthetic organic dye molecules, including indocyanine green, porphyrins coordinated with transition metals and naphthalocyanines, were externally used to enhance the photothermal effects, but not widely used in clinical settings as a result of the quick photobleaching of the dye molecules. Recently, gold metal at the nanoscale (billionths of a meter) has shown superior light absorption efficiency and the ability to convert photon energy into heat quickly and efficiently compared to conventional dye molecules, thereby making it a superior contrast agent for PTT. During the past few decades, a variety of nanostructures with unique optical properties for PTT have been characterized [70-73], and the plasmonic property of the metallic nanostructures is thus termed as plasmonic PTT (PPTT), which has become popular in recent years and has shown some therapeutic success [71]. Currently, the most promising nanostructures demonstrated in PPTT include gold nanospheres [74-77], gold nanorods [78-80] and gold nanoshells [81-84] for their strongly enhanced absorption in the visible and near-infrared (NIR) regions and their ease of preparation, multifunctionalization, and tunable optical properties. Other nanostructures include gold nanocages [85,86] and carbon nanotubes [87]. PPT exposes biological tissues to high temperatures to promote the destruction of tumor cells and, at the same time, excludes normal tissue from the process, thus making aptamers an excellent agent for conjugation to gold for specific targeting of neoplasms and simultaneously protecting normal tissue. Huang et al. [88] developed aptamer-conjugated Au-Ag nanorods for selective PTT in a cell mixture suspension for sharper and stronger longitudinal SPR compared to Au nanorods. In this study, the CCRF-CEM cell line was chosen as a 8

target and the NB-4 cell line was used as a control. Meanwhile, aptamer sgc8 with high affinity and specificity to the CCRFCEM cell line was chosen, and a random DNA library was used as a control. Results demonstrated that sgc8-conjugated Au-Ag nanorods exhibited cell-specific targeting after 5 min of irradiation with a laser light of 808 nm at 600 mW, leading to 90% cell death of CCRF-CEM cells and negligible cell death of NB-4 cells. These results indicate the capacity of this treatment to effectively destroy the target cancer cells with only minimal damage to the surrounding cells. Beqa et al. [89] developed an S6 aptamer-conjugated gold nanopopcorn-decorated single-wall carbon nanotube (GNPOP-SWNT) for selective PTT. Here, the HER2-positive SKBR3 breast cancer cell line was chosen as target and HER2-negative MDA-MB and HaCaT cell lines were used as control. GNPOP-SWNT targeted only HER2-positive SKBR3 breast cancer cells, resulting in effective PTT. Recently, Wang et al. [90] reported a strategy to modify gold nanorods with two different aptamers to destroy different cancer cells simultaneously. In this study, aptamer cancer stem cell (CSC)1 and aptamer CSC13 selected against DU145 prostate cancer cells and their subpopulation of CSCs, respectively, were conjugated to gold nanorods and successfully used to target and kill both DU145 cells and their subpopulation of CSCs under near-infrared laser irradiation (n = 3, p < 0.05). To further improve the photothermal efficacy of nanorods, Wu et al. [91] synthesized a novel Ag-Au nanostructure exhibiting a high capability of absorbing NIR radiation superior to that of Au-Ag NRs and modified it with the S2.2 aptamer that specifically bound to MUC1 for PTT. He found that the aptamer-conjugated Ag-Au NRs performed effective PTT of MCF-7 cells at a very low irradiation power density without destroying nontarget cells. Choi et al. [92] demonstrated a smart PTT agent, using EGFR aptamer (AptEGFR)-conjugated PEG-layered gold nanorods (AptEGFR-PGNRs). The optical properties, biocompatibility, colloidal stability, binding affinity and epithelial cancer cell killing efficacy in vitro and in vivo under NIR laser irradiation were investigated and showed the excellent tumor targeting ability of AptEGFR-PGNRs (n = 5, p < 0.05). Aptamer bioconjugates for targeted drug or siRNA delivery

6.2.6

In addition to the types described above, some other nanomaterials, including liposomes, quantum dots (QDs), and gold NPs, have also been used as aptamer conjugates for target drug delivery. Liposomes are self-assembled, spherically shaped structures composed of an outer lipid bilayer surrounding a central aqueous space. Kang et al. [93] developed a multifunctional target-specific delivery system based on aptamer sgc8 and liposomes. He mixed hydrogenated soy phosphatidylcholine, cholesterol, methoxy-poly-(ethyleneglycol)-distearoyl-phosphatidyl-ethanolamine, and maleimideterminated PEG-DSPE (Mal-PEG) in the ratio of

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Cell-specific aptamers and their conjugation with nanomaterials for targeted drug delivery

A.

B.

H N

O

C.

MUC1

C Quantum dot

CONHNH2

Au

CONHN OH

COOH C

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DOX

Lipid

Intercalated DOX

sgc8-TMR

PEG Dextan-FITC

Figure 5. (A) Schematic representation of quantum dot for drug delivery. (B) Schematic representation of gold nanoparticle for drug delivery. (C) Schematic representation of multifunctional liposome for drug delivery.

2:1:0.08:0.02 to construct liposomes (Figure 5). Fluorescein isothiocyanate--dextran was then encapsulated into the liposomes to serve as a model drug as well as reporter. The results showed that the liposomes could be internalized into targeted cells. Xing et al. [94] developed a liposomal drug delivery system functionalized with AS1411, a DNA aptamer with strong binding affinity for nucleolin, for targeted anticancer chemotherapy. AS1411 aptamer-functionalized liposomes containing Dox as a payload increased cellular internalization and cytotoxicity to MCF-7 breast cancer cells as well as improved antitumor efficacy against xenograft MCF-7 breast tumors in athymic nude mice compared to nontarget liposomes (n = 3, p < 0.01). A QD is a nanocrystal made of semiconductor materials whose excitons are confined in all three spatial dimensions. Bagalkot et al. [95] demonstrated that aptamers could simultaneously function as drug delivery and imaging agents. He used Dox and QD fluorescent molecules for this purpose and constructed a QD-aptamer-Dox conjugate, which, in the absence of target, presents quenching of both Dox and QD by a bi-fluorescence resonance energy transfer mechanism. However, when the QD-aptamer-Dox conjugate was internalized via PSMA-mediated endocytosis into prostate cancer cells, Dox was released, resulting in the recovery of fluorescence by both Dox and QD. The result demonstrated that QDaptamer-Dox was as cytotoxic as free Dox, but only minimally affected PSMA-negative PC3 cells, indicating the PSMAmediated specific targeting potential of QD-aptamer-Dox. Savla et al. [96] designed the delivery of a tumor-targeted, pH-responsive QD-MUC1 aptamer-Dox (QD-MUC1DOX) conjugate for the chemotherapy of ovarian cancer (Figure 5). The result showed that this conjugate had higher cytotoxicity than free DOX in multidrug-resistant cancer cells and preferentially accumulated in ovarian tumor (p < 0.05). The suspension of submicrometer-sized gold NPs (AuNPs) presents unique optical and electronic properties due to the size, shape and surface chemistry of AuNPs in solution. Recently, the unique optical-electronic properties of AuNPs

have been utilized in advanced biomedical and biological applications, including biosensors, therapeutic agents and drug delivery (Figure 5). Li et al. [97] first selected an RNA aptamer against EGFR and conjugated it with gold NPs. The experiment demonstrated that aptamer-coated gold NPs could specifically target and internalize EGFR-presenting A431 cells via EGFR-mediated endocytosis, indicating that these aptamer conjugates are suitable for carrying cargo molecules into EGFR-presenting cells. With their specific recognition ability, aptamers are also used to direct exogenous siRNAs that can silence gene expression via the RNAi pathway to target cells. McNamara et al. [98] reported a strategy that connects the PSMA aptamer A10 with therapeutic siRNAs specific for bcl-2 and plk1, two survival genes overexpressed in many human tumors, to bind PSMA on target cells via aptamer A10 and to silence the target mRNAs via the siRNA portions. The aptamer-siRNA chimeras resulted in target cell endocytosis, gene silencing, reduced cell proliferation and, finally, cell death, but without harming nontarget cells not presenting PSMA. Furthermore, the intratumoral application of the chimeras into mice carrying tumors derived from PSMA-positive human prostate cancer cells also showed a reduction of tumor growth, strongly suggesting that the cellular uptake of the aptamer was highly efficient and specific for PSMA (n = 8, p < 0.0001). Kim et al. [99] recently reported a strategy to kill cancer cells with a combinatorial approach. They constructed anti-PSMA aptamer-conjugated polyethylenimine and PEG to co-deliver Dox and small hairpin RNA against gene Bcl-xL to selectively and potently kill LNCaP cells in vitro. The results showed aptamer-conjugated polymers and NPs to be promising strategies for targeted drug delivery with minimal toxic side effects. Zhou et al. [15] selected a group of gp120 RNA atpamers that can specifically bind to the surface of cells expressing gp120 and were able to prevent the interaction between HIV and its receptor, allowing neutralization of HIV-1 infectivity. However, the extensive glycosylation of gp120 allows

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HIV to escape the immune response, thus compromising the effectiveness of the aptamers. They designed aptamer-siRNAchimeras for targeted delivery of antiviral (tat/rev) siRNA via gp160-mediated endocytosis. Results showed that the chimeras could specifically downregulate the mRNA levels of the HIV transcripts tat and rev [100]. One year later, they selected new 2¢-fluoropyrimidine-modified gp120-binding aptamers that blocked HIV-1 infection of cultured CEM T cells [15]. They again demonstrated the cell-specific delivery process with aptamer-siRNA-chimeras, resulting in successful downregulation of tat and rev mRNAs and the reduction of HIV-1 replication in PBMCs, suggesting that the antigp120 aptamers may be promising agents for an anti-HIV therapy by their ability to escort drugs into affected cells. 7.

Conclusion

Since the first introduction of cell-SELEX two decades ago, aptamers have attracted increasing interest and have been applied in biomedicine and bioanalysis to achieve multiple purposes based on their high affinity and specificity as well as facile modification compared with antibodies. Cell-SELEX is a promising technology because it selects aptamers against whole live cells without prior knowledge of resident proteins on the cell surface, making it an ideal tool for preferential binding to diseased cells, especially cancer cells, as well as biomarker discovery. Over the past two decades, this technology has enabled the generation of aptamers for a myriad of proteins, including PSMA, PTK7, IGHM, and nucleolin. Nanotechnology is an emerging field and a wide variety of NPs already exists. NPs possess many advantages over bulk structures and have already been widely used for drug delivery. Bioconjugation of NPs with aptamers exploits both technologies, making aptamer-NP conjugates ideal agents for drug delivery with proven therapeutic effects and the reduction of toxicity to normal tissue. 8.

Expert opinion

DNA or RNA aptamers with their high specificity and affinity for target cells can be easily conjugated with therapeutic agents for use in targeted drug delivery. Aptamers with high inhibitory potential, low molecular weight and low-to-no immunogenicity or toxicity, possess superior advantages as probes for targeted therapy compared with their counterpart antibodies, as well as comparable affinities with their target. The higher physicochemical stability and ease of chemical modification further enable aptamers to be engineered to improve their targeting potency and in vivo lifetimes. At the same time, their admirable optical, magnetic, electrochemical and mechanical properties make nanomaterials good candidates as signal generation and transduction components as well as delivery vehicles. Recent progress in the field of targeted drug delivery based on aptamers/nanomaterials bioconjugation has been achieved by taking advantage of the 10

characteristics of aptamers and nanomaterials as well as their integration with chemotherapeutic agents [101-105]. Meanwhile, the road ahead is long and many difficulties still require resolution. First, although all kinds of aptamers could theoretically be selected through SELEX technology, the selection procedure itself is time consuming and labor intensive, so further refinement of cell-SELEX techniques is needed to facilitate the identification of more disease targets and improve targeting efficiency. Second, because aptamers are NA, they are highly susceptible to degradation by several nucleases in vivo. Also, the small size of aptamers makes them liable to renal filtration. To address these problems, aptamers often are modified with different kinds of agents resulting in increasing cost and side effects. As shown above, the conjugation of aptamers and nanomaterials makes them ideal agents for targeted drug delivery and, among these nanomaterials, the emergence of micelles provides more promising future possibilities for drug delivery. However, instability is a problem. In addition, the conjugate should first be able to pass through the tumor blood vessels by passive processes of random diffusion and convectional flow. Once the conjugate successfully passes through the tumor blood vessels, it should be viable to attach to its specific target and release the drug at the tumor site. On the other hand, as the anti-tumor drugs reach the tumor and retard tumor growth or kill tumor cells, the tumor blood vessels may change and the blood flow that once attract aptamers to promote tumor targeting may decrease. Moreover, when tumor cells are killed, the remaining cells may mutate and present different markers on their surfaces. One method to address this problem may be the use of multivalent aptamers. For example, one kind of aptamer conjugated with nanomaterials may target the tumor cells whereas the other aptamer promotes immunocyte (such as T-cells) activation and generates an anti-tumor immune response to inhibit and kill the remaining tumor cells. Finally, the decoration of multivalent aptamers on nanomaterials for drug delivery to different cancer subtypes, multimerization of aptamers with drugs to increase drug loading capacity and development of facile conjugation strategies are all challenges that remain to be addressed. So far, only a modest amount of in vivo animal data has been confirmed and human data demonstrating aptamer-mediated delivery feasibility are still lacking. Meanwhile, the long-term side effects of NPs should be considered in the human body. Currently, however, many clinical trials are underway with various aptamer/nanomaterial assemblies, and the therapeutic efficacy of targeted delivery has already been improved. Thus, we anticipate that solving such shortcomings of aptamer/nanomaterial applications is a realistic goal for the near future.

Acknowledgements The authors wish to thank Prof. Weihong Tan for his discussion and help with this paper.

Expert Opin. Drug Deliv. (2014) 12(3)

Cell-specific aptamers and their conjugation with nanomaterials for targeted drug delivery

Declaration of interest This work was supported by the National Natural Science Foundation of China (NSFC 81370983), the National Key Scientific Instrument and Equipment Development Project (2011YQ03012414), the Hunan Provincial Science and

Technology Program (S2014S2032) and the Science and research foundation of Hunan health department (132011014). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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101. Wang R, Zhu G, Mei L, et al. Automated modular synthesis of aptamer-drug conjugates for targeted drug delivery. J Am Chem Soc 2014;136:2731-4 102. Zhu G, Zheng J, Song E, et al. Self-assembled, aptamer-tethered DNA nanotrains for targeted transport of molecular drugs in cancer theranostics. Proc Natl Acad Sci USA 2013;110:7998-8003 103. Yuan Q, Wu Y, Wang J, et al. Engineering a targeted bioimaging and photodynamic therapy nanoplatform using an aptamer-guided G-quadruplex DNA carrier with near-infrared light. Angew Chem Int Ed 2013;52:13965-9 104. Hu R, Zhang X, Zhao Z, et al. DNA nanoflowers for multiplexed cellular imaging and traceable targeted drug delivery. Angew Chem Int Ed 2014;53:5821-6 105. Zhao Z, Meng H, Wang H, et al. A nanocarrier with pHe-driven targeting and translocating for drug delivery. Angew Chem Int Ed 2013;52:7487-91

Affiliation Jie Liao1 PhD, Bo Liu2 PhD, Jun Liu2 PhD, Jiani Zhang1 PhD, Ke Chen3 PhD & Huixia Liu†4 PhD † Author for correspondence 1 Physician in the Department of Geriatric Medicine, Specializing in Endocrinology, Central South University, Xiang Ya Hospital, Changsha, China 2 Resident in the Department of Geriatric Medicine, Specializing in Endocrinology, Central South University, Xiang Ya Hospital, Changsha, China 3 Candidate, Majoring in Endocrinology and Geriatrics, Central South University, Xiang Ya Hospital, Changsha, China 4 Professor and Director, Department of Geriatric Medicine, Specialization in Endocrinology, Central South University, Xiang Ya Hospital, P.O. Box 190, Changsha, Hunan 410008, China Tel: +8613807483737; E-mail: [email protected]

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Expert Opin. Drug Deliv. (2014) 12(3)

Cell-specific aptamers and their conjugation with nanomaterials for targeted drug delivery.

Aptamers are short, single-stranded DNA or RNA sequences that can fold into complex secondary and tertiary structures and bind to various target molec...
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