CHEMMEDCHEM MINIREVIEWS DOI: 10.1002/cmdc.201402312
Nucleic Acid Aptamer-Mediated Drug Delivery for Targeted Cancer Therapy Huijie Zhu,[a] Jin Li,[a] Xiao-Bing Zhang,*[a] Mao Ye,[a] and Weihong Tan*[a, b] Aptamers are emerging as promising therapeutic agents and recognition elements. In particular, cell-SELEX (systematic evolution of ligands by exponential enrichment) allows in vitro selection of aptamers selective to whole cells without prior knowledge of the molecular signatures on the cell surface. The advantage of aptamers is their high affinitiy and binding specificity towards the target. This Minireview focuses on singlestranded (ss) oligonucleotide (DNA or RNA)-based aptamers as cancer therapeutics/theranostics. Specifically, aptamer–nano-
material conjugates, aptamer–drug conjugates, targeted phototherapy and targeted biotherapy are covered in detail. In reviewing the literature, the potential of aptamers as delivery systems for therapeutic and imaging applications in cancer is clear, however, major challenges remain to be resolved, such as the poorly understood pharmacokinetics, toxicity and offtarget effects, before they can be fully exploited in a clinical setting.
Introduction Today, cancer therapy is based primarily on chemotherapy and radiation, both of which have well-established side effects. Chemotherapy drugs typically kill not only cancer cells but also normal cells, and administration of bolus doses of these strong drugs can result in unpredictable side effects.[1] Moreover, the cell surface exhibits a host of proteins and other structures in a morphologically and physiologically complex microenvironment. However, for decades, the foundation of cancer diagnosis has rested on tumor cell or tissue morphology, instead of specifically targeting the key surface biomarkers. Even though genomics and proteomics have advanced the study of cells and tissues at the molecular level, methods that respond directly at the cell surface are still needed. One promising avenue can be found in aptamers, which can be both peptide and nucleic acid-based. Peptide aptamers are extremely simple combinatorial protein molecules in which a variable peptide sequence with affinity for a given target protein is displayed on an inert, constant scaffold protein.[2] Peptide aptamers are mainly used in medical therapy and in vivo diagnostics; several artificial combinatorial proteins are currently in preclinical studies and a few of them are in clinical trials.[2d, 3] However, this Minireview covers nucleic acid aptam[a] H. Zhu, J. Li, Prof. X.-B. Zhang, Prof. M. Ye, Prof. W. Tan Molecular Science & Biomedicine Laboratory State Key Laboratory of Chemo/Biosensing & Chemometrics College of Chemistry & Chemical Engineering, and College of Biology Collaborative Innovation Center for Molecular Engineering for Theranostics Hunan University, Changsha, 410082 (China) E-mail: [email protected] [b] Prof. W. Tan Center for Research at Bio/Nano Interface Departments of Chemistry and Physiology & Functional Genomics Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute University of Florida, Gainesville, FL 32611-7200 (USA) E-mail: [email protected]
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ers only, and for convenience, nucleic acid aptamer is abbreviated as aptamer in the remaining text. Nucleic acid aptamers are single-stranded (ss) oligonucleotide (DNA or RNA) molecules that fold into distinct secondary or tertiary structures, giving them high affinity and specific binding abilities toward their corresponding targets. Aptamers are selected from a random library of 1013–1016 ssDNA or ssRNA molecules through an in vitro technology known as SELEX (systematic evolution of ligands by exponential enrichment).[4] Aptamers are emerging as promising therapeutic agents and have been widely used as recognition elements with several advantages over antibodies, including more efficient and cost-effective chemical synthesis, easy and controllable modification with functional moieties to meet various clinical requirements, large-scale commercial production, excellent stability, nontoxicity, availability of antidotes for agonistic aptamers,[5] rapid tissue penetration, and limited immunogenicity.[6] They were first reported in the 1990s,[4] and since then, a large variety of aptamers has been developed with individual targets ranging from small molecules[4a, 7] and metal ions[7c] to proteins.[4b, 7c, 8] In 2006, the Tan group reported cell-based SELEX (cell-SELEX) by using CCRF-CEM, a cultured precursor Tcell acute lymphoblastic leukemia (ALL) cell line, as the target for in vitro selection. DNA aptamers generated from cell-SELEX target whole living cells, and they recognize the relatively native conformation of a certain target molecule on the cell membrane, very commonly a cell surface transmembrane protein.[9] Aptamers generated through cell-SELEX can be acquired without having prior knowledge of the molecular signatures of proteins found on the cell surface, making aptamers ideal tools for applications in cancer diagnosis and treatment. The three major steps of cell-SELEX, including incubation, partitioning and amplification, are shown in Figure 1. Using this method, ChemMedChem 0000, 00, 1 – 8
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Figure 1. Schematic illustration of the cell-SELEX (systematic evolution of ligands by exponential enrichment) process. DNA sequence specifically recognizing target cells is enriched and cloned. Positive clones are sequenced to identify individual aptamers. Copyright 2006 National Academy of Sciences. Reproduced with permission from Reference [12].
DNA aptamers against several kinds of cancer cells, such as lung cancer, liver cancer,[10] ovarian cancer,[11] ALL (T-cell leukemia),[12] colorectal cancer,[13] B-cell lymphoma,[14] breast cancer,[15] and acute myeloid leukemia (AML),[16] have been developed. Table 1 shows some examples of nucleic acid aptamers that bind to targets of therapeutic interest;[17] some other nucleic acid aptamers have been described in the literature.[18] In addition to their exceptional binding profile, aptamers have shown equally outstanding potential in biomarker discovery, cell sorting, detection,[7d] imaging,[19] and clinical treatment.[20] This Minireview focuses on aptamer-based cancer therapeutics/theranostics.
www.chemmedchem.org tumor imaging.[25] Moreover, they offer specific competitive advantages over antibodies and other protein biologics.[26] In 1990, Sullenger et al. first reported aptamers used as therapeutics.[27] In their work, overexpression of transactivation response (TAR)-containing sequences (TAR decoys) were used to render cells resistant to HIV replication. The TAR-containing sequences worked as aptamers to prevent the activation of viral gene expression, ultimately inhibiting viral replication. As another example, pegaptanib sodium (Macugen), a VEGF 165 aptamer, is regarded as the most commercially successful aptamer.[28] After direct injection into the vitreous cavity, Macugen binds to VEGF 165 and inhibits binding between VEGF 165 and its receptor, thus facilitating the treatment of age-related macular degeneration. Other examples include an anticoagulant aptamer that is able to inhibit thrombosis in murine models and systemically induce anticoagulation in pigs.[5a] Specifically, ARC1779,[29] as an aptamer drug, binds to the A1 domain of activated von Willebrand factor (vWF) with high affinity (Kd ~ 2 nm), leading to an antithrombotic effect, but without causing significant anticoagulation, on the basis of shear-dependent regulation of vWF function for arterial circulation. Another example can be found in the treatment of transmissible spongiform encephalopathies (TSEs), which are caused by prions: virus-like misfolded proteins. An RNA aptamer was identified that is able to bind to the prion protein PrPC and inhibit its conversion to PrPsc.[24, 30]
Aptamers for Targeted Drug Delivery With their high binding affinity and specificity toward cancer cells and associated proteins, aptamers can also be used to develop targeted drug delivery platforms. Targeted therapy can improve cancer treatment through increased efficacy, lower toxicity, and fewer side effects. In particular, the use of cellSELEX to generate multiple cancer-cell-specific aptamers has enabled researchers to make significant strides in developing methods of targeted therapy, such as chemotherapy and phototherapy.
Aptamers as Therapeutics Aptamers have a number of desirable characteristics when used as therapeutics, including high affinity and specificity, and biological efficacy. Excellent pharmacokinetic properties were observed in mice by Hicke et al., who used fluorescent molecules and TTA1 aptamers labeled with 99mTc to study in vivo
Targeted chemotherapy Chemotherapy is one of the most important therapeutic modalities. Unfortunately, because of their lack of specificity and tendency to induce cytotoxicity in both cancerous and healthy cells, chemotherapeutic drugs exhibit decreased efficacy and
Table 1. Examples of nucleic acid aptamers that bind to molecular targets of therapeutic interest. Aptamer
Molecular target(s)
Associated disease(s)
Refs.
Macugen AS1411 Sgc8 TD05 ARC1779 TBA
Vascular endothelial growth factor (VEGF) Nucleolin Protein tyrosine kinase 7 (PTK-7) Immunoglobulin m heavy chains (IGHM) A1 Domain of von Willebrand factor (vWF) a-Thrombin
Age-related macular degeneration Cancer Cancer Lymphoma Thrombotic microangiopathies and carotid artery disease Thrombosis
[21] [22] [23] [14] [24] [8a]
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increased side effects. However, aptamers generated from cellSELEX show high affinity and specificity for their cognate targets. As such, aptamer–nanomaterial conjugates and aptamers intercalated physically with drug are two drug delivery platforms that address the limitations of systemic chemotherapy. Aptamer–nanomaterial conjugates The unique properties of nanomaterials make them excellent drug carriers and signal reporters in cancer treatments. Indeed, aptamers conjugated with nanomaterials can improve the efficiency of cancer treatment, and various nanomaterials, such as gold and silica nanoparticles,[31] as well as carbon-based nanomaterials, including graphene or fullerenes, have been used to construct aptamer–nanomaterial conjugates for cancer treatment. For example, aptamer–Chlorin e6 (Ce6)-conjugated gold nanorods (AuNRs) could specifically bind to and kill targeted cells by photodynamic therapy (PDT) and photothermal therapy (PTT).[32] In a similar manner, a smart multifunctional nanostructure (SMN), composed of porous hollow magnetite nanoparticles capable of encapsulating doxorubicin (Dox), polyethylene glycol (PEG) ligands, and aptamers, could transport the SMN–Dox complexes into target cells and inhibit target cell proliferation.[33] As a kind of emerging nanomaterial, quantum dots garnered extensive investigation as potential drug delivery vehicles. Savla et al. reported the design and delivery of a tumor-targeted, pH-responsive quantum dot–mucin1 aptamer–doxorubicin (QD-MUC1-DOX) conjugate for the chemotherapy of ovarian cancer.[34] This system demonstrated the high potential of the proposed conjugate in the treatment of multidrug-resistant ovarian cancer. In recent years, DNA self-assembly has also been developed for targeted cancer therapy. The Tan group used base-pair hybridization to design a multifunctional aptamer-based DNA nanoassembly (AptNA) with various Y-shaped functional DNA domains, including targeting aptamers, intercalated anticancer drugs, and therapeutic antisense oligonucleotides.[35] As shown in Figure 2, the Y-shaped functional DNA domains were linked to an X-shaped DNA core connector through self-assembly and further photo-cross-linked into a multifunctional and programmable aptamer-based nanoassembly for targeted cancer therapy. This bulky nanoassembly provides many available sites for high-capacity loading of therapeutics or bioimaging agents. In addition, AptNAs show excellent biostability in a physiological environment (pH 7.4), thus avoiding unnecessary leaking of intercalated drugs during delivery. The aptamer–nanomaterial conjugates described above can only be used as drug carriers. In order to improve treatment, Wu et al. designed and engineered a further therapeutic platform. In particular, an aptamer–lipid–poly(lactide-co-glycolic acid) (PLGA) hybrid nanoparticle was synthesized with coreshell lipid-polymeric structures by simple nanoprecipitation and self-assembly.[36] Aptamer Sgc8, targeting human protein tyrosine kinase 7 (PTK7) overexpressed on CCRF-CEM cells, was hybridized with a diacyllipid-modified DNA strand, and DOX could be intercalated within the lipid–PEG–aptamer, Then, leci 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Schematic illustration of the multifunctional self-assembled nanoassembly building units and photo-cross-linked nanoassembly structure. Various Y-shaped functional DNA domains through predesigned base-pair hybridization were linked to an X-shaped DNA core connector, termed a building unit. Finally, ~ 100–200 of these basic building units with 5’-modification with acrydite groups were further photo-cross-linked into a multifunctional and programmable aptamer-based nanoassembly structure. Copyright 2013 American Chemical Society. Reproduced with permission from Reference [35].
thin, DSPE–PEG and lipid–PEG–aptamer loaded with DOX formed the hydrophilic shell. Thus, hydrophobic PLGA with encapsulated hydrophobic paclitaxel (PTX) composed the core structure, as shown in Figure 3.[36] Similarly, the Lu group has developed aptamer–liposome bioconjugates that can effectively deliver cisplatin in a cancer-cell-specific manner.[37] Their studies demonstrate that an aptamer-mediated cancer-targeting strategy is highly specific and can be modulated for desired drug delivery applications. In another example, acrydite was used to construct aptamer-tethered polymers by automated solid-phase DNA synthesis.[38] These aptamers were then able to polymerize in the presence of ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), allowing specific binding with, and internalization into, target cells, leading to cytotoxicity. Some clinical studies have shown that the combinatorial incorporation of DOX and PTX increases antitumor efficacy compared with the individual drugs,[39] and that these two drugs, even with different release rates, exhibit a synergistic effect. The cytotoxicity results also showed that this targeted co-delivery system selectively enhances antitumor efficacy.[40] Other nanomaterials include nanoflowers, which are self-assembled from long DNA building blocks generated via rolling circle replication (RCR) of a designer template, and micelles, which spontaneously self-assemble to form amphiphilic oligonucleotide molecules. Both can be used for targeted anticancer drug delivery integrated with aptamers and drug-loading sites.[41] Making full use of DNA nanoassembly not only improves the ability to identify a target cell but also provides an efficient method of drug delivery. As an example, the Tan group designed and developed a simple, target-specific, economical, and biocompatible drug delivery platform, termed aptamerChemMedChem 0000, 00, 1 – 8
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Figure 3. Schematic illustration of self-assembled hybrid nanoparticles for targeted co-delivery of two different drugs into cancer cells. Copyright 2014 Royal Society of Chemistry. Reproduced with permission from Reference [36].
tethered DNA nanotrains (aptNTrs), for targeted cancer therapy.[42] 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 decreased side effects in a mouse xenograft tumor model, making them promising targeted drug transport platforms for cancer theranostics. Aptamers modified with drugs for therapy Apart from aptamer–nanomaterial conjugates, aptamers have also been modified with cytotoxic agents, including Dox, docetaxel, daunorubicin, and cisplatin, or toxins, such as gelonin, to serve as drugs. It is anticipated that these aptamer–drug conjugates (ApDCs) will result in fewer side effects, while at the same time increasing therapeutic efficacy through the specific targeting by the aptamer. For example, the Tan group has studied the covalent linkage of Dox to DNA aptamer sgc8c.[43] This ApDC was able to specifically deliver the drug and kill targeted CCRF-CEM cells with minimal damage towards normal cells. While the method could effectively decrease the cytotoxicity of chemotherapy and increase its therapeutic effect, drug– aptamer crosslinking proved to be cumbersome. To address this problem, Wang et al. designed an automated modular synthesis of aptamer–drug conjugates for targeted drug delivery.[44] As shown in Figure 4, they designed a universal phosphoramidite containing an anticancer drug moiety and a photocleavable linker. By using this module, multiple drugs were efficiently incorporated into ApDCs at predesigned positions. As a result, the ApDCs achieved both specific recognition of 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 4. (A) Automated and modular synthesis of aptamer-drug conjugates from the phosphoramidites A, T, C, G, and D. (B) Structural features of phosphoramidite D. Copyright 2014 American Chemical Society. Reproduced with permission from Reference [44].
target cancer cells and the release of drugs in a photocontrollable manner. The results demonstrated the potential of automated and modular ApDC technology for applications in targeted cancer therapy. Targeted phototherapy PDT has emerged as an effective, noninvasive, and economical treatment for different malignancies. However, like chemotherapy, PDT also has some side effects, such as high internal temperature. In 2008, the Tan group engineered a novel molecular complex including a photosensitizer, Ce6, that was covalently attached to one end of the DNA aptamer, which wraps onto the surface of single-walled carbon nanotubes (SWNTs) for controllable singlet oxygen (1O2) generation.[45] Then, Huang et al. designed an aptamer-targeted PDT system[46] composed of three functional modules: a fluorescent photosensitizer Ce6, human angiogenin (ang), and a 45-mer ang aptamer. The aptamer was used as a linker between ang and Ce6. By using this model, they achieved specific and efficacious PDT. In order to improve the PDT effect, Han et al. designed and engineered an aptamer-based DNA nanocircuit[47] capable of selective recognition of cancer cells, controllable activation of photosensitizers, and amplification of the photodynamic therapeutic effect by using a catalyzed hairpin assembly (CHA). Current photosensitizers, such as Ce6, used for phototherapy need to be excited by UV light, which is an undesirable region of the wavelength spectrum for clinical applications. Therefore, to make phototherapy more practical, near-IR (NIR) excitation is being considered. In order to achieve this goal, the Tan group used up-conversion nanoparticles (UCNPs), which are able to emit short wavelength photons under excitation by NIR light, to implement an aptamer-guided G-quadruplex DNA ChemMedChem 0000, 00, 1 – 8
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nanoplatform for targeted PDT and bioimaging.[48] As shown in Figure 5, an aptamer is linked to a guanine-rich DNA segment to form a G4-aptamer and a bifunctional DNA sequence. The G4-aptamer, which is bioconjugated to a UCNP, serves as both a carrier for the photosensitizer TMPyP4, a kind of porphyrin derivative, and as a recognition element for target cells. When the nanoplatform is delivered into cancer cells, the UCNPs are excited by NIR light to emit visible light to image cancer cells. At the same time, the UCNPs initiate the photonic conversion of light to heat through the photosensitizer TMPyP4, which in turn generates cytotoxic reactive oxygen species (ROS). The UCNP–G4–aptamer–drug can be used as a platform for the development of many novel effective cancer chemotherapy methodologies.
Targeted biotherapy In addition to traditional cancer treatments, such as chemotherapy and phototherapy, scientists have developed new cancer treatments. These include aptamer-modified immune cells, whereby immune cells are redirected to kill cancer cells in vivo, while at the same time sparing normal cells. For example, natural killer lymphocytes (NK cells) are currently under study for direct treatment of cancer.[49] The Tan group has used two different aptamers, Sgc8 targeting PTK7 on the cell-surface membrane of CCRF-CEM cells and TD05 targeting IGHM on the surface of Ramos cells,[50] for testing. As shown in Figure 6, the aptamers are linked by a PEG segment to a synthetic diacyllipid tail with two stearic acids, which acts as the membrane anchor. Immune effector cells modified with aptamers were shown to recognize leukemia cells through major histocompatibility complex (MHC) nonrestricted structures, thus leading to elevated cancer cell targeting and killing. This was the first report of aptamer-controlled T-cell redirection to kill cancer cells with specificity.
Figure 6. Schematic illustration of targeting cancer cells (c) with aptamermodified immune cells (a). After incubating with lipo-aptamer probes (shown in center), immune cells recognize and kill cancer cells in the cell mixture, avoiding normal (b) cells. Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Adapted with permission from Reference [50].
Conclusions and Outlook
Since their discovery in the 1990s, aptamers, with their high affinity and specific targeting ability, have been widely used in analytical chemistry, biochemistry, medicine and several other fields. In this Minireview, we have highlighted the major applications of nucleic acid aptamers in the biomedical field based on cell-SELEX technology. Taking advantage of the specific recognition ability of these aptamers, scientists have developed aptamer–drug complexes and designed aptamer targeting systems against cancer cells. Some general engineering/design principles in developing such aptamer-based delivery systems include: no effect of modifications on the recognition capabilities of aptamers (no effect on aptamer conformation), release of the drug after entering the cell (by enzymatic degradation, reduction of disulfide bond, etc.), and the capability to carry a variety of possible drugs (such as DNA nanotrains).[42a] Such nanoplatforms can be used in chemotherapy, photodynamic therapy and biological therapy for cancer with facile modification, high stability and programmability.[43, 51] These methods have improved the efficiency of cancer treatment, while decreasing side effects. While targeted therapy using aptamer-based platforms is expected to play a significant role Figure 5. A targeted photodynamic therapy nanoplatform using an aptamer-guided G-quadruplex DNA carrier in the clinical treatment of and near-infrared irradiation. Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission cancer in the future, many of the from Reference [48]. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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CHEMMEDCHEM MINIREVIEWS engineering concepts described here work in the laboratory, but remain outside practical application in clinical settings. Some of these and other aptamers are now being tested in clinical trials and one has been approved by the US Food and Drug Administration (FDA).[52] Moreover, only a limited number of screened aptamer sequences related to diseases are currently available. Therefore, to design and engineer nucleic acid aptamers as chemical antibodies for application in more disease states, more aptamers need to be screened, and new therapeutic programs need to undergo clinical trials. Although aptamer-based delivery systems appear to hold potential for cancer therapy, major challenges and issues remain to be resolved, such as the poorly understood pharmacokinetics, toxicity and off-target effects.[17, 42b] To realize the full potential and overcome the challenges of such multifunctional nanosystems, it is necessary to perform more stringent in vivo testing to demonstrate the effectiveness of these systems. The continued development of animal models for the evaluation of safety and efficacy of these promising therapeutic strategies will lay the foundation for use in humans. Keywords: Aptamers · Cancer · Drug delivery · Nucleic acids · Theranostics · Therapeutics [1] L. Brannon-Peppas, J. O. Blanchette, Adv. Drug Delivery Rev. 2004, 56, 1649 – 1659. [2] a) P. Colas, B. Cohen, T. Jessen, I. Grishina, J. McCoy, R. Brent, Nature 1996, 380, 548 – 550; b) F. Hoppe-Seyler, I. Crnkovic-Mertens, E. Tomai, K. Butz, Curr. Mol. Med. 2004, 4, 529 – 538; c) R. Seigneuric, J. Gobbo, P. Colas, C. Garrido, Oncotarget 2011, 2, 557 – 561; d) M. Mascini, I. Palchetti, S. Tombelli, Angew. Chem. Int. Ed. 2012, 51, 1316 – 1332; Angew. Chem. 2012, 124, 1342 – 1360. [3] a) C. Borghouts, C. Kunz, B. Groner, Expert Opin. Biol. Ther. 2005, 5, 783 – 797; b) K. Butz, C. Denk, A. Ullmann, M. Scheffner, F. Hoppe-Seyler, Proc. Natl. Acad. Sci. USA 2000, 97, 6693 – 6697; c) C. Buerger, B. Groner, J. Cancer Res. Clin. Oncol. 2003, 129, 669 – 675; d) A. Skerra, Curr. Opin. Biotechnol. 2007, 18, 295 – 304. [4] a) A. D. Ellington, J. W. Szostak, Nature 1990, 346, 818 – 822; b) C. Tuerk, L. Gold, Science 1990, 249, 505 – 510. [5] a) C. P. Rusconi, J. D. Roberts, G. A. Pitoc, S. M. Nimjee, R. R. White, G. Quick, E. Scardino, W. P. Fay, B. A. Sullenger, Nat. Biotechnol. 2004, 22, 1423 – 1428; b) S. Oney, R. T. Lam, K. M. Bompiani, C. M. Blake, G. Quick, J. D. Heidel, J. Y.-C. Liu, B. C. Mack, M. E. Davis, K. W. Leong, Nat. Med. 2009, 15, 1224 – 1228. [6] Eyetech Study Group, Retina 2002, 22, 143 – 152. [7] a) R. R. Breaker, G. F. Joyce, Chem. Biol. 1995, 2, 655 – 660; b) T. Hermann, D. J. Patel, Science 2000, 287, 820 – 825; c) N. K. Navani, Y. Li, Curr. Opin. Chem. Biol. 2006, 10, 272 – 281; d) L. Qiu, C. Wu, M. You, D. Han, T. Chen, G. Zhu, J. Jiang, R. Yu, W. Tan, J. Am. Chem. Soc. 2013, 135, 12952 – 12955. [8] a) L. C. Bock, L. C. Griffin, J. A. Latham, E. H. Vermaas, J. J. Toole, nature 1992, 355, 564 – 566; b) C.-h. B. Chen, G. A. Chernis, V. Q. Hoang, R. Landgraf, Proc. Natl. Acad. Sci. USA 2003, 100, 9226 – 9231; c) D. A. Daniels, H. Chen, B. J. Hicke, K. M. Swiderek, L. Gold, Proc. Natl. Acad. Sci. USA 2003, 100, 15416 – 15421; d) P. Mallikaratchy, R. V. Stahelin, Z. Cao, W. Cho, W. Tan, Chem. Commun. 2006, 3229 – 3231; e) P. Parekh, Z. Tang, P. C. Turner, R. W. Moyer, W. Tan, Anal. Chem. 2010, 82, 8642 – 8649; f) J. Hu, J. Wu, C. Li, L. Zhu, W. Y. Zhang, G. Kong, Z. Lu, C. J. Yang, ChemBioChem 2011, 12, 424 – 430; g) M. Khati, M. Schman, J. Ibrahim, Q. Sattentau, S. Gordon, W. James, J. Virol. 2003, 77, 12692 – 12698; h) W. Tan, M. J. Donovan, J. Jiang, Chem. Rev. 2013, 113, 2842 – 2862. [9] S. Ohuchi, Biores. Open. Access. 2012, 1, 265 – 272. [10] D. Shangguan, L. Meng, Z. C. Cao, Z. Xiao, X. Fang, Y. Li, D. Cardona, R. P. Witek, C. Liu, W. Tan, Anal. Chem. 2008, 80, 721 – 728.
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CHEMMEDCHEM MINIREVIEWS [37] Z. Cao, R. Tong, A. Mishra, W. Xu, G. C. Wong, J. Cheng, Y. Lu, Angew. Chem. Int. Ed. 2009, 48, 6494 – 6498; Angew. Chem. 2009, 121, 6616 – 6620. [38] L. Yang, L. Meng, X. Zhang, Y. Chen, G. Zhu, H. Liu, X. Xiong, K. Sefah, W. Tan, J. Am. Chem. Soc. 2011, 133, 13380 – 13386. [39] a) F. Ahmed, R. I. Pakunlu, A. Brannan, F. Bates, T. Minko, D. E. Discher, J. Controlled Release 2006, 116, 150 – 158; b) D. L. Gustafson, A. L. Merz, M. E. Long, Cancer Lett. 2005, 220, 161 – 169. [40] Y. Cui, Q. Xu, P. K.-H. Chow, D. Wang, C.-H. Wang, Biomaterials 2013, 34, 8511 – 8520. [41] a) G. Zhu, R. Hu, Z. Zhao, Z. Chen, X. Zhang, W. Tan, J. Am. Chem. Soc. 2013, 135, 16438 – 16445; b) H. Liu, Z. Zhu, H. Kang, Y. Wu, K. Sefan, W. Tan, Chem. Eur. J. 2010, 16, 3791 – 3797; c) Y. Wu, K. Sefah, H. Liu, R. Wang, W. Tan, Proc. Natl. Acad. Sci. USA 2010, 107, 5 – 10; d) R. Hu, X. Zhang, Z. Zhao, G. Zhu, T. Chen, T. Fu, W. Tan, Angew. Chem. Int. Ed. 2014, 53, 5821 – 5826; Angew. Chem. 2014, 126, 5931 – 5936. [42] a) G. Zhu, J. Zheng, E. Song, M. Donovan, K. Zhang, C. Liu, W. Tan, Proc. Natl. Acad. Sci. USA 2013, 110, 7998 – 8003; b) Q. Liu, C. Jin, Y. Wang, X. Fang, X. Zhang, Z. Chen, W. Tan, NPG Asia Mater. 2014, 6, e95. [43] Y. F. Huang, D. Shangguan, H. Liu, J. A. Phillips, X. Zhang, Y. Chen, W. Tan, ChemBioChem 2009, 10, 862 – 868. [44] R. Wang, G. Zhu, L. Mei, Y. Xie, H. Ma, M. Ye, F. L. Qing, W. Tan, J. Am. Chem. Soc. 2014, 136, 2731 – 2734. [45] Z. Zhu, Z. Tang, J. A. Phillips, R. Yang, H. Wang, W. Tan, J. Am. Chem. Soc. 2008, 130, 10856 – 10857. [46] X. Yang, J. Huang, K. Wang, W. Li, L. Cui, X. Li, ChemMedChem 2011, 6, 1778 – 1780.
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www.chemmedchem.org [47] D. Han, G. Zhu, C. Wu, Z. Zhu, T. Chen, X. Zhang, W. Tan, ACS Nano 2013, 7, 2312 – 2319. [48] Q. Yuan, Y. Wu, J. Wang, D. Lu, Z. Zhao, T. Liu, X. Zhang, W. Tan, Angew. Chem. Int. Ed. 2013, 52, 13965 – 13969; Angew. Chem. 2013, 125, 14215 – 14219. [49] a) M. J. Smyth, Y. Hayakawa, K. Takeda, H. Yagita, Nat. Rev. Cancer 2002, 2, 850 – 861; b) P. A. Albertsson, P. H. Basse, M. Hokland, R. H. Goldfarb, J. F. Nagelkerke, U. Nannmark, P. J. Kuppen, Trends Immunol. 2003, 24, 603 – 609. [50] X. Xiong, H. Liu, Z. Zhao, M. B. Altman, D. Lopez-Colon, C. J. Yang, L.-J. Chang, C. Liu, W. Tan, Angew. Chem. Int. Ed. 2013, 52, 1472 – 1476; Angew. Chem. 2013, 125, 1512 – 1516. [51] a) P. Mallikaratchy, H. Liu, Y. F. Huang, H. Wang, D. Lopez-Colon, W. Tan, Chem. Commun. 2009, 3056 – 3058; b) P. Mallikaratchy, Z. Tang, W. Tan, ChemMedChem 2008, 3, 425 – 428; c) A. Boltz, B. Piater, L. Toleikis, R. Guenther, H. Kolmar, B. Hock, J. Biol. Chem. 2011, 286, 21896 – 21905; d) P. R. Mallikaratchy, A. Ruggiero, J. R. Gardner, V. Kuryavyi, W. F. Maguire, M. L. Heaney, M. R. McDevitt, D. J. Patel, D. A. Scheinberg, Nucleic Acids Res. 2011, 39, 2458 – 2469; e) S. M. Douglas, I. Bachelet, G. M. Church, Science 2012, 335, 831 – 834. [52] a) X. Ni, M. Castanares, A. Mukherjee, S. E. Lupold, Curr. Med. Chem. 2011, 18, 4206 – 4214; b) X. Pei, J. Zhang, J. Liu, Mol. Clin. Oncol. 2014, 2, 341 – 348; c) P. Sundaram, H. Kurniawan, M. E. Byrne, J. Wower, Eur. J. Pharm. Sci. 2013, 48, 259 – 271. Received: July 25, 2014 Published online on && &&, 0000
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MINIREVIEWS H. Zhu, J. Li, X.-B. Zhang,* M. Ye, W. Tan* && – && Nucleic Acid Aptamer-Mediated Drug Delivery for Targeted Cancer Therapy
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Targeted & Specific: The applications of nucleic acid aptamers in cancer are reviewed. Single-stranded (ss) oligonucleotide (DNA or RNA)-based aptamers conjugated with drugs and nanomaterials are covered in detail, highlighting their therapeutic potential while acknowledging the challenges that remain to be overcome.
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Nucleic Acid Aptamer-Mediated Drug Delivery for Targeted Cancer Therapy Huijie Zhu,[a] Jin Li,[a] Xiao-Bing Zhang,*[a] Mao Ye,[a] and Weihong Tan*[a, b] Aptamers are emerging as promising therapeutic agents and recognition elements. In particular, cell-SELEX (systematic evolution of ligands by exponential enrichment) allows in vitro selection of aptamers selective to whole cells without prior knowledge of the molecular signatures on the cell surface. The advantage of aptamers is their high affinitiy and binding specificity towards the target. This Minireview focuses on singlestranded (ss) oligonucleotide (DNA or RNA)-based aptamers as cancer therapeutics/theranostics. Specifically, aptamer–nano-
material conjugates, aptamer–drug conjugates, targeted phototherapy and targeted biotherapy are covered in detail. In reviewing the literature, the potential of aptamers as delivery systems for therapeutic and imaging applications in cancer is clear, however, major challenges remain to be resolved, such as the poorly understood pharmacokinetics, toxicity and offtarget effects, before they can be fully exploited in a clinical setting.
Introduction Today, cancer therapy is based primarily on chemotherapy and radiation, both of which have well-established side effects. Chemotherapy drugs typically kill not only cancer cells but also normal cells, and administration of bolus doses of these strong drugs can result in unpredictable side effects.[1] Moreover, the cell surface exhibits a host of proteins and other structures in a morphologically and physiologically complex microenvironment. However, for decades, the foundation of cancer diagnosis has rested on tumor cell or tissue morphology, instead of specifically targeting the key surface biomarkers. Even though genomics and proteomics have advanced the study of cells and tissues at the molecular level, methods that respond directly at the cell surface are still needed. One promising avenue can be found in aptamers, which can be both peptide and nucleic acid-based. Peptide aptamers are extremely simple combinatorial protein molecules in which a variable peptide sequence with affinity for a given target protein is displayed on an inert, constant scaffold protein.[2] Peptide aptamers are mainly used in medical therapy and in vivo diagnostics; several artificial combinatorial proteins are currently in preclinical studies and a few of them are in clinical trials.[2d, 3] However, this Minireview covers nucleic acid aptam[a] H. Zhu, J. Li, Prof. X.-B. Zhang, Prof. M. Ye, Prof. W. Tan Molecular Science & Biomedicine Laboratory State Key Laboratory of Chemo/Biosensing & Chemometrics College of Chemistry & Chemical Engineering, and College of Biology Collaborative Innovation Center for Molecular Engineering for Theranostics Hunan University, Changsha, 410082 (China) E-mail: [email protected] [b] Prof. W. Tan Center for Research at Bio/Nano Interface Departments of Chemistry and Physiology & Functional Genomics Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute University of Florida, Gainesville, FL 32611-7200 (USA) E-mail: [email protected]
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ers only, and for convenience, nucleic acid aptamer is abbreviated as aptamer in the remaining text. Nucleic acid aptamers are single-stranded (ss) oligonucleotide (DNA or RNA) molecules that fold into distinct secondary or tertiary structures, giving them high affinity and specific binding abilities toward their corresponding targets. Aptamers are selected from a random library of 1013–1016 ssDNA or ssRNA molecules through an in vitro technology known as SELEX (systematic evolution of ligands by exponential enrichment).[4] Aptamers are emerging as promising therapeutic agents and have been widely used as recognition elements with several advantages over antibodies, including more efficient and cost-effective chemical synthesis, easy and controllable modification with functional moieties to meet various clinical requirements, large-scale commercial production, excellent stability, nontoxicity, availability of antidotes for agonistic aptamers,[5] rapid tissue penetration, and limited immunogenicity.[6] They were first reported in the 1990s,[4] and since then, a large variety of aptamers has been developed with individual targets ranging from small molecules[4a, 7] and metal ions[7c] to proteins.[4b, 7c, 8] In 2006, the Tan group reported cell-based SELEX (cell-SELEX) by using CCRF-CEM, a cultured precursor Tcell acute lymphoblastic leukemia (ALL) cell line, as the target for in vitro selection. DNA aptamers generated from cell-SELEX target whole living cells, and they recognize the relatively native conformation of a certain target molecule on the cell membrane, very commonly a cell surface transmembrane protein.[9] Aptamers generated through cell-SELEX can be acquired without having prior knowledge of the molecular signatures of proteins found on the cell surface, making aptamers ideal tools for applications in cancer diagnosis and treatment. The three major steps of cell-SELEX, including incubation, partitioning and amplification, are shown in Figure 1. Using this method, ChemMedChem 0000, 00, 1 – 8
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Figure 1. Schematic illustration of the cell-SELEX (systematic evolution of ligands by exponential enrichment) process. DNA sequence specifically recognizing target cells is enriched and cloned. Positive clones are sequenced to identify individual aptamers. Copyright 2006 National Academy of Sciences. Reproduced with permission from Reference [12].
DNA aptamers against several kinds of cancer cells, such as lung cancer, liver cancer,[10] ovarian cancer,[11] ALL (T-cell leukemia),[12] colorectal cancer,[13] B-cell lymphoma,[14] breast cancer,[15] and acute myeloid leukemia (AML),[16] have been developed. Table 1 shows some examples of nucleic acid aptamers that bind to targets of therapeutic interest;[17] some other nucleic acid aptamers have been described in the literature.[18] In addition to their exceptional binding profile, aptamers have shown equally outstanding potential in biomarker discovery, cell sorting, detection,[7d] imaging,[19] and clinical treatment.[20] This Minireview focuses on aptamer-based cancer therapeutics/theranostics.
www.chemmedchem.org tumor imaging.[25] Moreover, they offer specific competitive advantages over antibodies and other protein biologics.[26] In 1990, Sullenger et al. first reported aptamers used as therapeutics.[27] In their work, overexpression of transactivation response (TAR)-containing sequences (TAR decoys) were used to render cells resistant to HIV replication. The TAR-containing sequences worked as aptamers to prevent the activation of viral gene expression, ultimately inhibiting viral replication. As another example, pegaptanib sodium (Macugen), a VEGF 165 aptamer, is regarded as the most commercially successful aptamer.[28] After direct injection into the vitreous cavity, Macugen binds to VEGF 165 and inhibits binding between VEGF 165 and its receptor, thus facilitating the treatment of age-related macular degeneration. Other examples include an anticoagulant aptamer that is able to inhibit thrombosis in murine models and systemically induce anticoagulation in pigs.[5a] Specifically, ARC1779,[29] as an aptamer drug, binds to the A1 domain of activated von Willebrand factor (vWF) with high affinity (Kd ~ 2 nm), leading to an antithrombotic effect, but without causing significant anticoagulation, on the basis of shear-dependent regulation of vWF function for arterial circulation. Another example can be found in the treatment of transmissible spongiform encephalopathies (TSEs), which are caused by prions: virus-like misfolded proteins. An RNA aptamer was identified that is able to bind to the prion protein PrPC and inhibit its conversion to PrPsc.[24, 30]
Aptamers for Targeted Drug Delivery With their high binding affinity and specificity toward cancer cells and associated proteins, aptamers can also be used to develop targeted drug delivery platforms. Targeted therapy can improve cancer treatment through increased efficacy, lower toxicity, and fewer side effects. In particular, the use of cellSELEX to generate multiple cancer-cell-specific aptamers has enabled researchers to make significant strides in developing methods of targeted therapy, such as chemotherapy and phototherapy.
Aptamers as Therapeutics Aptamers have a number of desirable characteristics when used as therapeutics, including high affinity and specificity, and biological efficacy. Excellent pharmacokinetic properties were observed in mice by Hicke et al., who used fluorescent molecules and TTA1 aptamers labeled with 99mTc to study in vivo
Targeted chemotherapy Chemotherapy is one of the most important therapeutic modalities. Unfortunately, because of their lack of specificity and tendency to induce cytotoxicity in both cancerous and healthy cells, chemotherapeutic drugs exhibit decreased efficacy and
Table 1. Examples of nucleic acid aptamers that bind to molecular targets of therapeutic interest. Aptamer
Molecular target(s)
Associated disease(s)
Refs.
Macugen AS1411 Sgc8 TD05 ARC1779 TBA
Vascular endothelial growth factor (VEGF) Nucleolin Protein tyrosine kinase 7 (PTK-7) Immunoglobulin m heavy chains (IGHM) A1 Domain of von Willebrand factor (vWF) a-Thrombin
Age-related macular degeneration Cancer Cancer Lymphoma Thrombotic microangiopathies and carotid artery disease Thrombosis
[21] [22] [23] [14] [24] [8a]
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increased side effects. However, aptamers generated from cellSELEX show high affinity and specificity for their cognate targets. As such, aptamer–nanomaterial conjugates and aptamers intercalated physically with drug are two drug delivery platforms that address the limitations of systemic chemotherapy. Aptamer–nanomaterial conjugates The unique properties of nanomaterials make them excellent drug carriers and signal reporters in cancer treatments. Indeed, aptamers conjugated with nanomaterials can improve the efficiency of cancer treatment, and various nanomaterials, such as gold and silica nanoparticles,[31] as well as carbon-based nanomaterials, including graphene or fullerenes, have been used to construct aptamer–nanomaterial conjugates for cancer treatment. For example, aptamer–Chlorin e6 (Ce6)-conjugated gold nanorods (AuNRs) could specifically bind to and kill targeted cells by photodynamic therapy (PDT) and photothermal therapy (PTT).[32] In a similar manner, a smart multifunctional nanostructure (SMN), composed of porous hollow magnetite nanoparticles capable of encapsulating doxorubicin (Dox), polyethylene glycol (PEG) ligands, and aptamers, could transport the SMN–Dox complexes into target cells and inhibit target cell proliferation.[33] As a kind of emerging nanomaterial, quantum dots garnered extensive investigation as potential drug delivery vehicles. Savla et al. reported the design and delivery of a tumor-targeted, pH-responsive quantum dot–mucin1 aptamer–doxorubicin (QD-MUC1-DOX) conjugate for the chemotherapy of ovarian cancer.[34] This system demonstrated the high potential of the proposed conjugate in the treatment of multidrug-resistant ovarian cancer. In recent years, DNA self-assembly has also been developed for targeted cancer therapy. The Tan group used base-pair hybridization to design a multifunctional aptamer-based DNA nanoassembly (AptNA) with various Y-shaped functional DNA domains, including targeting aptamers, intercalated anticancer drugs, and therapeutic antisense oligonucleotides.[35] As shown in Figure 2, the Y-shaped functional DNA domains were linked to an X-shaped DNA core connector through self-assembly and further photo-cross-linked into a multifunctional and programmable aptamer-based nanoassembly for targeted cancer therapy. This bulky nanoassembly provides many available sites for high-capacity loading of therapeutics or bioimaging agents. In addition, AptNAs show excellent biostability in a physiological environment (pH 7.4), thus avoiding unnecessary leaking of intercalated drugs during delivery. The aptamer–nanomaterial conjugates described above can only be used as drug carriers. In order to improve treatment, Wu et al. designed and engineered a further therapeutic platform. In particular, an aptamer–lipid–poly(lactide-co-glycolic acid) (PLGA) hybrid nanoparticle was synthesized with coreshell lipid-polymeric structures by simple nanoprecipitation and self-assembly.[36] Aptamer Sgc8, targeting human protein tyrosine kinase 7 (PTK7) overexpressed on CCRF-CEM cells, was hybridized with a diacyllipid-modified DNA strand, and DOX could be intercalated within the lipid–PEG–aptamer, Then, leci 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Schematic illustration of the multifunctional self-assembled nanoassembly building units and photo-cross-linked nanoassembly structure. Various Y-shaped functional DNA domains through predesigned base-pair hybridization were linked to an X-shaped DNA core connector, termed a building unit. Finally, ~ 100–200 of these basic building units with 5’-modification with acrydite groups were further photo-cross-linked into a multifunctional and programmable aptamer-based nanoassembly structure. Copyright 2013 American Chemical Society. Reproduced with permission from Reference [35].
thin, DSPE–PEG and lipid–PEG–aptamer loaded with DOX formed the hydrophilic shell. Thus, hydrophobic PLGA with encapsulated hydrophobic paclitaxel (PTX) composed the core structure, as shown in Figure 3.[36] Similarly, the Lu group has developed aptamer–liposome bioconjugates that can effectively deliver cisplatin in a cancer-cell-specific manner.[37] Their studies demonstrate that an aptamer-mediated cancer-targeting strategy is highly specific and can be modulated for desired drug delivery applications. In another example, acrydite was used to construct aptamer-tethered polymers by automated solid-phase DNA synthesis.[38] These aptamers were then able to polymerize in the presence of ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), allowing specific binding with, and internalization into, target cells, leading to cytotoxicity. Some clinical studies have shown that the combinatorial incorporation of DOX and PTX increases antitumor efficacy compared with the individual drugs,[39] and that these two drugs, even with different release rates, exhibit a synergistic effect. The cytotoxicity results also showed that this targeted co-delivery system selectively enhances antitumor efficacy.[40] Other nanomaterials include nanoflowers, which are self-assembled from long DNA building blocks generated via rolling circle replication (RCR) of a designer template, and micelles, which spontaneously self-assemble to form amphiphilic oligonucleotide molecules. Both can be used for targeted anticancer drug delivery integrated with aptamers and drug-loading sites.[41] Making full use of DNA nanoassembly not only improves the ability to identify a target cell but also provides an efficient method of drug delivery. As an example, the Tan group designed and developed a simple, target-specific, economical, and biocompatible drug delivery platform, termed aptamerChemMedChem 0000, 00, 1 – 8
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Figure 3. Schematic illustration of self-assembled hybrid nanoparticles for targeted co-delivery of two different drugs into cancer cells. Copyright 2014 Royal Society of Chemistry. Reproduced with permission from Reference [36].
tethered DNA nanotrains (aptNTrs), for targeted cancer therapy.[42] 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 decreased side effects in a mouse xenograft tumor model, making them promising targeted drug transport platforms for cancer theranostics. Aptamers modified with drugs for therapy Apart from aptamer–nanomaterial conjugates, aptamers have also been modified with cytotoxic agents, including Dox, docetaxel, daunorubicin, and cisplatin, or toxins, such as gelonin, to serve as drugs. It is anticipated that these aptamer–drug conjugates (ApDCs) will result in fewer side effects, while at the same time increasing therapeutic efficacy through the specific targeting by the aptamer. For example, the Tan group has studied the covalent linkage of Dox to DNA aptamer sgc8c.[43] This ApDC was able to specifically deliver the drug and kill targeted CCRF-CEM cells with minimal damage towards normal cells. While the method could effectively decrease the cytotoxicity of chemotherapy and increase its therapeutic effect, drug– aptamer crosslinking proved to be cumbersome. To address this problem, Wang et al. designed an automated modular synthesis of aptamer–drug conjugates for targeted drug delivery.[44] As shown in Figure 4, they designed a universal phosphoramidite containing an anticancer drug moiety and a photocleavable linker. By using this module, multiple drugs were efficiently incorporated into ApDCs at predesigned positions. As a result, the ApDCs achieved both specific recognition of 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 4. (A) Automated and modular synthesis of aptamer-drug conjugates from the phosphoramidites A, T, C, G, and D. (B) Structural features of phosphoramidite D. Copyright 2014 American Chemical Society. Reproduced with permission from Reference [44].
target cancer cells and the release of drugs in a photocontrollable manner. The results demonstrated the potential of automated and modular ApDC technology for applications in targeted cancer therapy. Targeted phototherapy PDT has emerged as an effective, noninvasive, and economical treatment for different malignancies. However, like chemotherapy, PDT also has some side effects, such as high internal temperature. In 2008, the Tan group engineered a novel molecular complex including a photosensitizer, Ce6, that was covalently attached to one end of the DNA aptamer, which wraps onto the surface of single-walled carbon nanotubes (SWNTs) for controllable singlet oxygen (1O2) generation.[45] Then, Huang et al. designed an aptamer-targeted PDT system[46] composed of three functional modules: a fluorescent photosensitizer Ce6, human angiogenin (ang), and a 45-mer ang aptamer. The aptamer was used as a linker between ang and Ce6. By using this model, they achieved specific and efficacious PDT. In order to improve the PDT effect, Han et al. designed and engineered an aptamer-based DNA nanocircuit[47] capable of selective recognition of cancer cells, controllable activation of photosensitizers, and amplification of the photodynamic therapeutic effect by using a catalyzed hairpin assembly (CHA). Current photosensitizers, such as Ce6, used for phototherapy need to be excited by UV light, which is an undesirable region of the wavelength spectrum for clinical applications. Therefore, to make phototherapy more practical, near-IR (NIR) excitation is being considered. In order to achieve this goal, the Tan group used up-conversion nanoparticles (UCNPs), which are able to emit short wavelength photons under excitation by NIR light, to implement an aptamer-guided G-quadruplex DNA ChemMedChem 0000, 00, 1 – 8
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nanoplatform for targeted PDT and bioimaging.[48] As shown in Figure 5, an aptamer is linked to a guanine-rich DNA segment to form a G4-aptamer and a bifunctional DNA sequence. The G4-aptamer, which is bioconjugated to a UCNP, serves as both a carrier for the photosensitizer TMPyP4, a kind of porphyrin derivative, and as a recognition element for target cells. When the nanoplatform is delivered into cancer cells, the UCNPs are excited by NIR light to emit visible light to image cancer cells. At the same time, the UCNPs initiate the photonic conversion of light to heat through the photosensitizer TMPyP4, which in turn generates cytotoxic reactive oxygen species (ROS). The UCNP–G4–aptamer–drug can be used as a platform for the development of many novel effective cancer chemotherapy methodologies.
Targeted biotherapy In addition to traditional cancer treatments, such as chemotherapy and phototherapy, scientists have developed new cancer treatments. These include aptamer-modified immune cells, whereby immune cells are redirected to kill cancer cells in vivo, while at the same time sparing normal cells. For example, natural killer lymphocytes (NK cells) are currently under study for direct treatment of cancer.[49] The Tan group has used two different aptamers, Sgc8 targeting PTK7 on the cell-surface membrane of CCRF-CEM cells and TD05 targeting IGHM on the surface of Ramos cells,[50] for testing. As shown in Figure 6, the aptamers are linked by a PEG segment to a synthetic diacyllipid tail with two stearic acids, which acts as the membrane anchor. Immune effector cells modified with aptamers were shown to recognize leukemia cells through major histocompatibility complex (MHC) nonrestricted structures, thus leading to elevated cancer cell targeting and killing. This was the first report of aptamer-controlled T-cell redirection to kill cancer cells with specificity.
Figure 6. Schematic illustration of targeting cancer cells (c) with aptamermodified immune cells (a). After incubating with lipo-aptamer probes (shown in center), immune cells recognize and kill cancer cells in the cell mixture, avoiding normal (b) cells. Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Adapted with permission from Reference [50].
Conclusions and Outlook
Since their discovery in the 1990s, aptamers, with their high affinity and specific targeting ability, have been widely used in analytical chemistry, biochemistry, medicine and several other fields. In this Minireview, we have highlighted the major applications of nucleic acid aptamers in the biomedical field based on cell-SELEX technology. Taking advantage of the specific recognition ability of these aptamers, scientists have developed aptamer–drug complexes and designed aptamer targeting systems against cancer cells. Some general engineering/design principles in developing such aptamer-based delivery systems include: no effect of modifications on the recognition capabilities of aptamers (no effect on aptamer conformation), release of the drug after entering the cell (by enzymatic degradation, reduction of disulfide bond, etc.), and the capability to carry a variety of possible drugs (such as DNA nanotrains).[42a] Such nanoplatforms can be used in chemotherapy, photodynamic therapy and biological therapy for cancer with facile modification, high stability and programmability.[43, 51] These methods have improved the efficiency of cancer treatment, while decreasing side effects. While targeted therapy using aptamer-based platforms is expected to play a significant role Figure 5. A targeted photodynamic therapy nanoplatform using an aptamer-guided G-quadruplex DNA carrier in the clinical treatment of and near-infrared irradiation. Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission cancer in the future, many of the from Reference [48]. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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MINIREVIEWS H. Zhu, J. Li, X.-B. Zhang,* M. Ye, W. Tan* && – && Nucleic Acid Aptamer-Mediated Drug Delivery for Targeted Cancer Therapy
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Targeted & Specific: The applications of nucleic acid aptamers in cancer are reviewed. Single-stranded (ss) oligonucleotide (DNA or RNA)-based aptamers conjugated with drugs and nanomaterials are covered in detail, highlighting their therapeutic potential while acknowledging the challenges that remain to be overcome.
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These are not the final page numbers! ÞÞ
