Aptamers: an emerging navigation tool of therapeutic agents for targeted cancer therapy

Chang Yang ab, Yu Jiang ac, Sai Heng Hao d, Xing Yi Yan bc, De Fei Hong *e and Hua Naranmandura *abcf
aDepartment of Hematology, the First Affiliated Hospital, and Department of Public Health, Zhejiang University School of Medicine, Hangzhou, China. E-mail: narenman@zju.edu.cn
bDepartment of Toxicology, School of Medicine and Public Health, Zhejiang University, Hangzhou, China
cDepartment of Pharmacology, School of Medicine, Zhejiang University, Hangzhou, China
dCollege of Pharmaceutical Sciences, Inner Mongolia Medical University, Hohhot, China
eDepartment of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China. E-mail: hongdefi@163.com
fZhejiang University Cancer Center, Hangzhou, China

Received 25th September 2021 , Accepted 21st November 2021

First published on 22nd November 2021


Abstract

Chemotherapeutic agents have been used for the treatment of numerous cancers, but due to poor selectivity and severe systemic side effects, their clinical application is limited. Single-stranded DNA (ssDNA) or RNA aptamers could conjugate with highly toxic chemotherapy drugs, toxins, therapeutic RNAs or other molecules as novel aptamer–drug conjugates (ApDCs), which are capable of significantly improving the therapeutic efficacy and reducing the systemic toxicity of drugs and have great potential in clinics for targeted cancer therapy. In this review, we have comprehensively discussed and summarized the current advances in the screening approaches of aptamers for specific cancer biomarker targeting and development of the aptamer–drug conjugate strategy for targeted drug delivery. Moreover, considering the huge progress in artificial intelligence (AI) for protein and RNA structure predictions, automatic design of aptamers using deep/machine learning techniques could be a powerful approach for rapid and precise construction of biopharmaceutics (i.e., ApDCs) for application in cancer targeted therapy.


1. Introduction

Cancer is a leading cause of death worldwide, and current major treatment approaches for cancer management include surgery, radiation therapy, chemotherapy, targeted therapy, hormone therapy and immunotherapy.1–3 Among them, chemotherapy, either alone or in combination with surgery or radiation therapy, is the most widely used therapeutic approach.1,2 In fact, chemotherapy agent induced cancer cell death is predominantly dependent on its powerful cytotoxicity by inhibiting the process of cell mitosis or cell division. Thus, it not only kills cancer cells, but also affects other normal cells with rapidly dividing properties, resulting in severe side effects as well as decreasing the overall quality-of-life for patients and therapeutic efficacy.4–6

In recent years, targeted therapy, which uses drugs or other substances to precisely attack cancer cells with little damage to normal cells, has grown rapidly.6,7 Clinically, tyrosine kinase inhibitor Imatinib used in the treatment of Philadelphia chromosome positive leukemia through specific inhibition of BCR-ABL tyrosine kinase has led to the opening of a new area for the investigation of cancer treatment drugs.8,9 Currently, there are two major types of targeted therapy drugs, namely, small molecule drugs and monoclonal antibodies (mAb)10–12 Particularly, monoclonal antibodies could precisely recognize tumor specific surface antigens (e.g., receptors), block their downstream cell signalling pathways, and finally suppress cancer cell proliferation. Moreover, mAbs can also be used to deliver high toxic payloads (e.g., chemotherapy drugs or toxins) for precisely killing cancer cells, which can dramatically reduce their severe side effects.11,12 Since the first antibody–drug conjugate (ADC) Mylotarg was approved by FDA for the treatment of CD33 positive acute myeloid leukemia (AML) patients, scientists have applied more than fifty ADCs for clinical trials.11 However, ADCs also have certain limitations, such as high cost, low stability due to their protein nature, notable inter batch variability and high immunogenicity.13–15 Therefore, other types of high affinity molecules are needed as a supplement to ADCs for targeted therapy of cancer.

Aptamers are short, single-stranded DNA or RNA molecules that are able to recognize target molecules through their three-dimensional (3D) conformation with high binding affinity.16,17 Since Ellington et al. and Tuerk et al. reported for the first time an in vitro method that could screen RNA species specifically recognizing their ligands, aptamers have been rapidly developed for broad applications such as diagnosis, biosensors and clinical therapy.18–20 Pegaptanib sodium (Macugen), an RNA aptamer targeting vascular endothelial growth factor (VEGF) with high binding affinity, has been used in the treatment of wet age-related macular degeneration.21 Similar to antibodies, aptamers are able to serve as not only therapeutic drugs for targeted inhibition of tumor related signalling pathways, but also as drug carriers (i.e., aptamer–drug conjugates, ApDCs) to precisely deliver therapeutic agents such as chemotherapeutic drugs, therapeutic RNAs, toxins and radioisotopes into cancer cells or tumors.22 In reality, ApDCs offer some unique advantages over ADCs, for instance, aptamer selection is often faster and less expensive than antibody generation, and aptamers have high thermal and chemical stability, and low immunogenicity, undergo chemical modification easily and penetrate tissues rapidly (Table 1).23–25 In this review, we have primarily focused on the different strategies and the latest advances in the construction of aptamer-based drug delivery systems for targeted therapy.

Table 1 Comparison of the characteristics of ApDCs and ADCs
Characteristics ApDCs ADCs
Molecular weight Low High
Manufacturing Simple (chemical synthesis) Complicated (in vivo immunization + chemical synthesis)
Batch variation Low High
Immunogenicity Low High
Chemical stability Stable Sensitive to pH value and temperature
Chemical modification Easy Difficult
Tissue penetration Rapid Slow
Cost Low High
Clearance Rapid Slow


2. Screening of aptamers with drug delivery potential

Different from the antibody production through utilizing the immunization of animals, aptamers are usually generated by an in vitro selection method called Systematic Evolution of Ligands by Exponential enrichment (SELEX), in which aptamers are screened from a randomized ssDNA or RNA library by an iterative process after several selection rounds.18–20 Thereby, the technology for screening aptamers is extremely important for obtaining high binding affinity aptamers (i.e., Kd values ranging from nM to pM) that can target cancer cell surface antigens and precisely deliver drugs into cells.26 Currently, protein-based, cell-based and animal model-based SELEX approaches are frequently used to select the aptamers with drug delivery potential (Table 2). Here, we have described and summarized the recent advances in the SELEX technology.
Table 2 Comparison of the advantages and disadvantages between three SELEX approaches
Protein-based SELEX Cell-based SELEX In vivo SELEX
Model Purified proteins Live cells CDX or PDX models
Target All types of proteins Membrane protein Membrane protein
Advantages Wide range of target Recognition of target with native form; without the prior knowledge about target proteins High biostability; high specificity; suitable for cancer metastasis model; capable of crossing the blood–brain barrier
Disadvantages Time consuming; non-specificity; failure on recognition of target with natural conformation Time consuming; failure for in vivo use High cost


2.1 Membrane protein-based SELEX approach

It is well known that aptamers are capable of precisely delivering drugs into cancer cells by targeting cell surface antigens. Generally, purified recombinant proteins from prokaryotic systems are commonly used as targets for aptamer selection in vitro, which is named protein-based SELEX.27 For instance, in the protein-based SELEX approach, a randomized ssDNA (or RNA) library is mixed with target proteins for specific binding enrichment, while the unbound sequences are discarded through various solid-phase matrix-based segmentation strategies such as nitrocellulose membranes, columns, magnetic beads, and capillary electrophoresis,27,28 as shown in Fig. 1a. Moreover, the bound sequences are then separated from their targets and amplified by PCR to generate an enriched pool for the next round of aptamer selection until the ssDNA or RNA pools reached the highest binding affinity. Several aptamers targeting specific antigens have been identified by the above-mentioned approach, and certain aptamers were applied to clinical trials for the treatment of disease.29,30
image file: d1tb02098f-f1.tif
Fig. 1 Current common SELEX approaches for selection of high binding affinity aptamers. (A) The procedure of protein-based SELEX for aptamer selection: (1) incubate ssDNA library with the purified membrane protein target; (2) discard unbound aptamers and isolate the aptamers that bind to target proteins; (3) additionally, incubate the target bound aptamers with non-target proteins (e.g., albumin); (4) after incubation, discard the albumin bound aptamers and collect and amplify unbound aptamers by PCR as an enriched pool for the next round of selection. After a few rounds of selection, high-throughput sequencing is performed for sequence analysis. (B) Aptamer selection procedure of cell-based SELEX: (1) incubate ssDNA library with targeted positive cells; (2) discard unbound aptamers and (3) isolate the aptamers that bind to positive cells; (4) isolated aptamers from positive cells are further incubated with negative cells; (5) discard negative cell bound aptamers and amplify the unbound aptamers by PCR as the enriched pool for the next round of selection. After a few rounds of selection, high-throughput sequencing is performed for sequence analysis. (C) Procedure of Hybrid-SELEX: the enriched pool from cell-based SELEX (as described in B) is further applied to protein-based SELEX (as described in A) to obtain high binding affinity aptamers, and then high-throughput sequencing is performed for sequence analysis. (D) In vivo-SELEX for aptamer selection: (1) transplantation of tumor cells into immunodeficient mice to construct a CDX model; (2) Intravenous injection of ssDNA library into the CDX mouse model; (3) harvest tumor tissue and (4) isolate the aptamers from the tumor tissue; (5) amplify isolated aptamers by PCR and finally obtaining sequence information through high-throughput sequencing. Created with http://BioRender.com.

However, it was also pointed out that certain recombinant proteins purified from a prokaryotic expression system cannot fold into an exact structure due to the lack of protein posttranslational modifications such as phosphorylation and glycosylation in eukaryotic systems.28 Consequently, screened aptamers sometimes are unable to recognize and interact with the epitope of their corresponding targets in live cells, resulting in failure of biomedical application. Moreover, due to lack of knowledge about cancer specific antigens for certain types of cancers such as CD19 negative leukemia,31,32 it is unable to screen aptamers targeting such cancer cells using the protein-based SELEX approach, suggesting that the recombinant protein-based approach also has some limitations.

2.2 Cell-based SELEX approach

Considering the shortcoming of protein-based SELEX, target protein expressed cells are employed for aptamer screening, which is called cell-based SELEX. In this method, live cells which highly express target membrane receptors are utilized as positive cells for aptamer screening, and the oligonucleotide pools (i.e., RNA or DNA) are directly incubated with positive cells for obtaining cell bound sequences (Fig. 1b).33,34 However, it is worth noting that the presence of other untargeted proteins on the cell surface would result in the off-target selection. Thereby a counter selection using negative cells without expression of target proteins is introduced in the cell-based SELEX approach to eliminate off-target sequences. Compared with the protein-based SELEX approach, the cell-based SELEX approach can shorten the selection procedure and increase the reliability of the selected aptamers. Moreover, selected aptamers from the classic cell-based SELEX approach also frequently failed to recognize their prescribed targets, which is actually attributed to the membrane protein expression profile difference between the negative and positive cells for aptamer selection.35 In other words, the difference in the membrane protein expression between positive and negative cells contains not only target proteins, but also other non-target proteins, leading to screened aptamers targeting undesired membrane proteins.36 For instance, the use of Daudi cells (CD38 positive) and HL-60 cells (CD38 negative) for the selection of CD38 targeted aptamers may lead to acquisition of aptamers targeting CD19, because CD19 is equally positively and negatively expressed in Daudi and HL-60 cells, respectively.

In order to avoid getting unspecific aptamers, scientists constructed the modified cell-based SELEX approach in which the same cell line is employed for negative and positive selections; for instance, cell lines overexpressing target proteins are produced by gene transfection as positive selection cells, and the parental mock cells are used for counter selection.36 By using this approach, aptamers targeting myeloid differentiation antigen CD33 as well as cancer stem cell marker CD133 with high affinity and specificity have been successfully obtained.36,37 In this study, CD33 transfected HEK293T cells are used as positive selection cells for aptamer enrichment, while the parental HEK293T cells (i.e., without transfection) are used as negative selection cells.36 A random ssDNA library is incubated with CD33-negative HEK293T cells in 6-well plates for 6 time negative selection, and the unbound ssDNA in the supernatant is further incubated with CD33-overexpressed HEK293T cells for positive selection. This simple approach could rapidly obtain reliable, stable and high binding affinity aptamers targeting antigens with only three rounds of selection and skillfully solved the above uncertainties.

Moreover, Hicke's Lab has introduced a hybrid-SELEX approach that combines the cell-based SELEX approach with the protein-based SELEX approach to avoid the off-target selection.38 Herein, after certain rounds of classical cell-SELEX, the selected oligonucleotide pool is used as the initial pool for additional rounds of selection with recombinant proteins as a crossover SELEX. The subsequent rounds of selection with target proteins are primarily carried out to enrich the high affinity aptamers and ensure that the selected aptamers are able to bind with target proteins.

On the other hand, it is important to note that oligonucleotide pools are incubated with cells below 4 °C during the classical cell SELEX approach, while the receptor mediated endocytosis could not be induced under this condition.39 Thus, the candidates selected from sequencing results should be further tested whether they could induce endocytosis after binding with their targets at 37 °C. Conversely, another modified cell-based SELEX approach, termed the cell-internalization SELEX, is designed to directly select functional aptamers that can be internalized into target cells.40 In this approach, oligonucleotide pools are incubated with positive selection cells at 37 °C, unbound aptamers as well as aptamers that remained on the cell surface are removed with a stringent wash step, and the internalized sequences are isolated, then amplified by PCR for the generation of an enriched aptamer pool. Using this method, Tanaka et al. selected aptamers that can specifically recognize non-small-cell lung cancer A549 cells and precisely deliver MALAT1-targeting antisense oligonucleotides into the inside of cells via endocytosis.41

2.3 In vivo-based SELEX approach

Here, a few important issues are concerned. First, screening of aptamers by an in vitro SELEX system (e.g., protein or cell based SELEX) is performed under simpler conditions, which is quite different from the complex physiological environment. Thereby the screened aptamers from in vitro selection cannot be guaranteed to be exactly functional in vivo. Second, in vitro selected aptamers (DNA or RNA) may be degraded by nucleases in the bloodstream before reaching the tumor region. Third, the tumor microenvironment that covers the tumors might prevent in vitro selected aptamers penetrating into tumor tissue.28 Therefore, a novel model that could simulate the in vivo environment during aptamer transportation is needed to improve the reliability of the selected aptamers for in vivo use.

In the last decade, the animal model-based (in vivo) SELEX approach is widely accepted for aptamer screening (Fig. 1c). For instance, ssDNA or RNA libraries are injected intravenously into a cell line-derived tumor xenograft (CDX) mouse model, and then these aptamers are isolated from the tumor tissues. The recovered sequences are further amplified for the next round of selection in vivo. After repeating the selection rounds a few times, the isolated aptamers are capable of recognizing and penetrating into tumors in vivo. This technique has been used to identify several aptamers that could penetrate into the brain, bone metastasis prostate cancer cells or intrahepatic colorectal cancer metastases.42 On the other hand, different from antibody generation through in vivo immunization induced by known antigens, aptamers could be selected using the in vivo SELEX approach without prior knowledge of cancer specific antigens.26 Thus, animal-based SELEX is deemed to be the most direct and appropriate approach for screening aptamers. Notably, patient-derived xenograft (PDX) models, in which tumor fragments surgically dissected from cancer patients are directly transplanted into immunodeficient mice, have been used for personalized drug screening. Thereby, PDX models could also be used to select suitable aptamers for patient personalized therapy, which offers a new direction for increasing the clinical potential of aptamers.

3. Aptamer-based targeted drug delivery systems

Due to the lack of specificity, chemotherapy agents exhibited significant toxicity on non-cancerous cells (i.e., healthy cells), resulting in severe side effects, limiting their tolerance and effectiveness.5,6,43,44 Clinical data have indicated that blood forming cells in the bone marrow, hair follicles and the nervous system are most likely to be attacked by chemotherapeutic agents.45 Therefore, targeted delivery systems for chemotherapeutic agents need to be developed for preventing damage to healthy cells, increasing the drug efficacy on tumors and decreasing the side effects.

Here, we have highlighted the aptamers as drug carriers that can conjugate with chemotherapeutic agents or powerful marine toxins and precisely deliver them into tumors through targeting the specific cell surface antigens, and could dramatically improve the therapeutic effect of drugs in cancer treatment, as shown in Fig. 2a. Compared with ADCs, aptamers could be conjugated with drugs in the ratio range from 1[thin space (1/6-em)]:[thin space (1/6-em)]1 to 1[thin space (1/6-em)]:[thin space (1/6-em)]50, which is much higher than the drug-to-antibody ratio range (1[thin space (1/6-em)]:[thin space (1/6-em)]1–1[thin space (1/6-em)]:[thin space (1/6-em)]8), indicating that ApDCs could achieve higher therapeutic efficacy than ADCs.46 Thereby, we discussed a few chemotherapeutic agents or toxins which can be conjugated with a variety of aptamers for targeted therapy of different cancers.


image file: d1tb02098f-f2.tif
Fig. 2 Aptamer-based targeted drug delivery system. (A) Scheme illustration of aptamer delivery of anticancer drugs: (1) aptamer–drug conjugates recognize and bind with the target membrane protein of cancer cells, and (2) then internalized by target mediated endocytosis; after internalization, aptamer–drug conjugates are further transported from the endosomes (3) to lysosomes (4), and finally the anticancer drug is released from ApDC and exerts its anticancer activity. (B) Scheme of aptamer delivery of therapeutic RNAs for cancer treatment: (I) aptamer–RNAs conjugates first bind with the target membrane protein of cancer cells, and (II) then internalized by target mediated endocytosis; additionally, endosome containing aptamer–RNA conjugates (III) release the therapeutic RNAs into the cytoplasm (IV), and the released RNAs (V) are incorporated into RNA-induced silencing complexes (RISCs) to (VI) impair the target mRNAs. Created with http://BioRender.com.

3.1 Doxorubicin (anthracycline antibiotic)

Doxorubicin (Dox) is an anthracycline antibiotic isolated from the Streptomyces peucetius species. As one of the most common chemotherapeutic drugs, Dox is widely used for the treatment of a variety of malignancies such as leukemia, lymphoma, myeloma, breast cancer, prostate cancer, and others.47 Dox can intercalate into the base pairs of DNA's double helix (dsDNA) as well as bind to DNA-associated enzymes, ultimately resulting in DNA damage that leads to apoptosis in cancer cells through inhibiting the cell cycle. However, Dox also showed multidirectional side effects including low white and red blood cell counts, or dose dependent cardiotoxicity.47–49

Importantly, due to the presence of flat aromatic rings, Dox could directly intercalate into the double-stranded DNA through non-covalent interaction.50 Thus, Dox also deemed to be able to intercalate into aptamers as the stem regions of aptamers also contain double stranded structures (Fig. 3a). Using fluorescence quenching properties, it has been confirmed that aptamers and Dox could form a physical complex after incubation.50 Recently, several aptamer–Dox conjugates have been synthesized by using this simple method. For example, Liu et al. and Hu et al. constructed aptamer–Dox conjugates that can specifically recognize and deliver Dox into the HER-2 and MUC-1 positive tumor cells, respectively, finally inducing the cell death.51,52


image file: d1tb02098f-f3.tif
Fig. 3 Scheme of examples of various aptamer conjugates. (a) Aptamer–doxorubicin (Dox) conjugates by physical intercalation. (b) Conjugation of aptamers with chemotherapy drugs or toxins through a chemical linker. (c) Dual aptamer with Dox. (d) Nucleobase analogue incorporated aptamers. (e) Aptamer coated nanoparticles containing drugs or RNAs. (f) Aptamer–RNA conjugates. (g) Aptamer–radioisotope conjugates. Created with http://BioRender.com.

More interestingly, since Dox preferentially binds to repeated CG rich double-stranded 5′-GC-3′ or 5′-CG-3′ sequences, CG cargo which contains 10–16 base pair CG repeated sequences is commonly used for the linkage with aptamers as drug-intercalating sites to improve the capacity of Dox loading. Zhu et al. designed aptamer-tethered DNA nanotrains (aptNTrs), where a long CG repetitive dsDNA was linked at the 5′-end of a protein tyrosine kinase 7 (PTK7) targeted aptamer Sgc8. The aptamer nanotrains Sgc8-NTr exhibited high payload capacity for Dox, and the Sgc8-NTr–Dox conjugate showed potent antitumor activity and reduced the side effects in vivo.53

However, the number of payloads is not controllable on physical conjugation, therefore chemical synthesis is used to control the payloads of aptamer–Dox conjugates. Huang et al. chemically linked Dox with an aptamer (e.g., Sgc8) through an acid-labile hydrazone linker (Fig. 3b). This linker could control the loading capacity of Dox, and this conjugate can be precisely internalized into CEM cells, and release drugs from the aptamer–Dox conjugate in endosomes, ultimately inducing cell death.54 On the other hand, bi-specific aptamers, which are developed from two monovalent aptamers targeting different antigens, could give a broader range of recognition capabilities for the aptamer (Fig. 3c). Zhu et al. constructed a bi-specific aptamer (named SD), namely, aptamers sgc8c and sgd5a that target CEM and Toledo cells (respectively) are connected through a self-resemble dsDNA linker.55 Additionally, this bi-specific aptamer SD could physically conjugate with Dox at a molar ratio of 0.12, and the bi-aptamer–Dox conjugate dramatically induces both CEM and Toledo cell death in the cell mixture, indicating that the bi-specific aptamer allowed drug cytotoxicity to be specifically directed to more subtypes of cancer cells, which can sidestep the problem of cancer heterogeneity mediated drug resistance.

3.2 Gemcitabine and 5-FU (pyrimidine analogue family)

Therapeutic nucleobase analogues represent such a reasonable class of chemotherapy drugs used for the treatment of disease over several decades.56 Gemcitabine, a deoxycytidine analog, has been used for the treatment of various solid tumors and certain lymphoma clinically.57 As for the treatment of pancreatic cancer, gemcitabine has showed satisfactory results on improving the overall survival (OS), performance status and pain control. Although gemcitabine is now utilized as a standard treatment for pancreatic cancer, its therapeutic efficacy is still low.58 One possible reason for the resistance of gemcitabine in clinics is associated with the low uptake by pancreatic cancer cells. The uptake of gemcitabine by cancer cells is found to be mainly mediated by nucleoside transporters such as human equilibrative nucleoside transporter 1 (hENT1) and cation-dependent human concentrative nucleoside transporters 1/3 (hCNT1/3).58 Recent clinic studies further demonstrated that pancreatic cancer patients with low expression of hENT1 and hCNT3 had significantly worse prognosis after gemcitabine treatment, as compared to patients with high nucleoside transporters levels,59,60 indicating a strong correlation between the efficiency of transportation and drug efficacy of gemcitabine.

On the other hand, due to gemcitabine having a similar structure to that of natural nucleotides, it can be internally incorporated into DNA using solid phase DNA synthesis techniques.61 Park, et al. synthesized a gemcitabine contained aptamer (denoted as APTA-12) through substitution of a single guanine residue in a nucleolin targeted aptamer AS1411 with a gemcitabine phosphoramidite, and it could specifically target and accumulate in nucleolin positive pancreatic cancer cells, resulting in effectively suppressed cell proliferation in vitro and in vivo.62 More interestingly, gemcitabine can also be incorporated into RNA aptamers through transcription reactions catalyzed by special RNA polymerase. Sousa et al. identified a mutant T7 RNA polymerase (Y639F), which efficiently utilizes not only the ribonucleoside triphosphates (rNTPs) but also deoxyribonucleoside triphosphates (dNTPs)63 Likewise, Ray et al. successfully synthesized an EGFR targeted aptamer-gemcitabine polymer (Gem-E07 polymer) through enzymatic reactions by taking advantage of mutant T7 RNA polymerase (Fig. 3d), in which seven cytosine sites of aptamer E07 were actually enzymatically replaced by gemcitabine monophosphates.64 Moreover, Gem-E07 conjugates could strongly inhibit the growth of EGFR positive pancreatic cancer cells after internalization through clathrin-mediated endocytosis.

Similarly, another nucleobase analogue 5-fluorouracil (5-FU), a chemotherapy agent, is also used for incorporation into aptamers. Using the automated solid-phase DNA synthesis, 5 copies of 5-FU linked phosphoramidite were site-specifically loaded onto PTK targeted aptamer sgc8, which has proven to be highly effective for delivering 5-FU into PTK expressed HCT116 cells, finally inducing cell death.65 Additionally, Sven et al. incorporated 5-fluoro-2′-deoxyuridine (5-FUdR) into aptamer AIR-3 by T7 RNA polymerase variant Y639F. Surprisingly, the modified aptamer AIR-3-FdU still has comparable binding affinity to its parent aptamer AIR-3 targeting interleukin-6 receptor (IL-6R), which is capable of specifically releasing 5-FUdR into cancer cells after internalization, leading to cell cycle arrest in the S phase and inhibition of cell proliferation.66 Overall, these exciting technologies provide a new area for rapidly synthesizing aptamers containing nucleobase analogues for the treatment of different cancers.

3.3 Paclitaxel (plant alkaloids)

Paclitaxel (PTX), a diterpenoid organic compound separated from the bark and needles of the pacific yew tree, is one of the most common chemotherapy agents used for cancer therapy.67 As a microtubule spindle dynamics suppressor, PTX could bind to the N-terminal of the β-tubulin subunit of the tubulin α/β heterodimers that assemble to form microtubules, increasing its polymerization, and eventually inhibiting cell mitosis as well as inducing cell apoptosis.68 However, due to its poor water solubility, PTX is needed to be formulated in a lipid-based solvent polyoxyl castor oil and dehydrated ethanol before clinical use, and these two hydrotropic agents caused severe histamine-mediated hypersensitivity reactions in vivo.69 In addition to this, owing to its indiscriminative distribution in tumor and normal tissues, PTX frequently causes other serious side effects.70 Therefore, it is highly desirable to develop PTX derivatives with high water solubility as well as tumor-targeting properties.

In recent years, nanotechnology is used to improve the bioavailability of poorly soluble drugs in clinics, and so PTX is conjugated with nanoparticles to improve its water solubility.71 Nevertheless, the therapeutic efficacy of PTX containing nanoparticles is still unsatisfactory due to the lack of specificity, while the aptamer-functionalized nanoparticle PTX conjugates could overcome the shortcoming of its nanoparticles. Interestingly, when the nanoparticles containing PTX is conjugated with aptamers targeting MUC-1 cancer cells, it could precisely recognize MUC-1 positive cancer cells and target the delivery of PTX into cells (Fig. 3e)72 Furthermore, heparanase (HPA), an endoglycosidase that cleaves heparan sulfate, is highly expressed in numerous cancer cells and has shown to be involved in the growth, angiogenesis and metastasis of tumors. Duan et al. synthesized HPA-targeted aptamers with PTX-encapsulated PEGylated PLGA nanoparticle complexes (Apt-PTX-NP) to target breast cancer cells. As anticipated, Apt-PTX-NP not only enhanced the anti-invasive effect and exhibited superior anti-angiogenesis activity of PTX, but also inhibited the positive tumor growth significantly.73

PTX could also be directly conjugated with aptamers through a chemical linker without nanoparticles. Zhang et al. used a cathepsin B-labile dipeptide linker to synthesize a nucleolin (Ncu) targeted aptamer–PTX (NcuA–PTX) conjugate for targeting human ovarian cancer cells. The linker mediated NcuA–PTX conjugate has dramatically improved the solubility of PTX itself, and enhanced its uptake into cells (or tumor) through nucleolin mediated macropinocytosis in vitro as well as in vivo.74

3.4 Marine drugs MMAE and MMAF (marine toxins)

Marine toxins are poisons produced by various organisms ranging from small microbes to fish and snails.75 Among them, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), which are derivatives from auristatin, are widely used for conjugation with monoclonal antibodies to synthesize ADCs.75 In 2011, brentuximab vedotin (Adcentris), an anti-CD30 antibody-MMAE drug, was approved by the FDA for the treatment of Hodgkin's lymphoma and systemic anaplastic large-cell lymphoma.76

Similar to the synthesis strategy of ADCs, aptamers can also be conjugated with marine toxins through a chemical linker. Kratschmer et al. successfully synthesized MMAE and MMAF with aptamers targeting the epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) respectively through a cathepsin-cleavable valine citrulline linker. Both conjugates could selectively inhibit the proliferation of pancreatic cancer cells, ultimately inducing cell death.77 Moreover, Yoon et al. also constructed the conjugation of pancreatic ductal adenocarcinoma (PDAC) targeted aptamers with MMAE to yield the ApDCs, which have shown superior anticancer effect in vitro.78 Taken together, the conjugation of aptamers with chemotherapy drugs or marine toxins is shown to be a powerful strategy tool in targeted cancer therapy.

4. Delivery of therapeutic RNA by aptamers

RNA interference (RNAi) is a biological process by which short RNAs, including small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and microRNAs (miRNAs), could hybridize with a complement sequence in endogenous messenger RNA (mRNA), leading to the translational repression or degradation of target genes.79,80 Recently, RNAi-based therapies targeting tumor-related signalling pathways provide a feasible alternative option for cancer treatment. In 2018, the first RNA drug Onpatto was approved by the FDA for the treatment of polyneuropathy through formulation as a lipid complex injection.81 However, there are some limitations about the stability and half-life of RNAs in body fluids and organs owing to the presence of nucleases. Moreover, due to the large size and hydrophilic properties, it is difficult for therapeutic RNAs to diffuse passively through cell membranes into target cells.82,83 Thus, novel delivery systems (e.g., aptamers) could provide solutions to the challenges for RNA therapeutics and widen their therapeutic window.

4.1 Small interfering RNAs (siRNAs)

Small interfering RNAs (siRNAs) are short RNA sequences composed of a duplex of 20–24 nucleotides that could interact with the RNA-induced silencing complex (RISC) for gene silencing.84 In 2006, Chu et al. for the first time constructed an aptamer–siRNA chimera consisting of a prostate-specific membrane antigen (PSMA) targeting RNA aptamers and anti-lamin A/C (or anti-GAPDH) siRNAs. For instance, both the aptamer and siRNAs are first biotinylated, and then aptamer–siRNA chimera is formed through non-covalent binding of biotin with streptavidin protein.85 Moreover, this aptamer–siRNA chimera effectively silenced the target genes in PSMA positive cells at similar levels to that obtained by transfection in vitro, while it had no effect on PSMA negative cells. Similarly, McNamara II et al. designed and synthesized a chimeric RNA containing a PSMA targeted aptamer and siRNA targeting polo-like kinase 1 (PLK1) or Bcl-2 (Fig. 3f). This chimeric RNA could specifically suppress their targeted genes by siRNA in PSMA-expressing cancer cells through aptamers, leading to the reduction of cell proliferation as well as tumor progression in vitro and in vivo.86

On the other hand, conjugation of two different types of siRNAs simultaneously with aptamers is performed for dual-targeting of oncogenic pathways in prostate cancer cells.87 In this study, siRNAs silencing survivin and EGFR are conjugated to the bivalent PSMA-targeted aptamer, and the results show that this chimera effectively suppressed the endogenous expression of both EGFR and survivin in the cells. In addition, in C4-2 prostate cancer cell-xenograft mice, the tumor growth was also significantly suppressed, demonstrating that delivery of two different types of siRNAs by bivalent aptamers is a more effective therapeutic strategy for treating cancers.

4.2 MicroRNAs (miRNAs)

MicroRNAs (miRNAs) are a group of small non-coding RNAs (19–24 nt) and are associated with the regulation of tumorigenesis and drug resistance.88,89 Besides, it has recently been reported that certain microRNAs play a critical role in tumor suppression as well. Thus, tumor suppressor miRNAs are interesting candidates as therapeutic RNAs in cancer treatment.90 Esposito et al. engineered an aptamer–miRNA chimera by fusing passenger strands of tumor suppressor let-7g miRNA targeting HMGA2 to the 3′ end of the aptamer (GL21.T) which recognizes oncogenic receptor tyrosine kinase Axl on cancer cell membranes with high affinity and specificity. As anticipated, this aptamer–miRNA chimera could specifically deliver the miRNA let-7g in Axl positive non-small cell lung cancer A549 cells as well as A549 cell derived tumors, resulting in inhibition of tumor growth significantly by down-regulation of HMGA2 genes.91 Meanwhile, Rohde et al. generated a sticky bridge connected chimera consisting of a TfA-targeted aptamer and pre-miR-126 to specifically inhibit the expression of vascular cell adhesion molecule-1 (VCAM-1) protein in TfA positive cancer cells. This conjugate dramatically reduced the proliferation of MCF-7 cancer cells, and decreased paracrine endothelial cell recruitment by silencing the VCAM-1 gene, indicating that targeted delivery of tumor suppressor miRNAs by aptamers could also be a useful approach for cancer therapy.92

4.3 Short hairpin RNAs (shRNAs)

Short hairpin RNAs (shRNAs) are RNA molecules with a hairpin loop structure and with a mechanism of action based on RNA interference-mediated post-transcriptional gene silencing of target genes.93 Recently, shRNA has been applied in many fields, and research is still ongoing in new areas of focus. Askarian et al. reported a targeted delivery of Bcl-xL shRNA by aptamer functionalized nanoparticles, in which the nucleolin DNA aptamer (AS1411) is conjugated to poly(L-lysine)-modified polyethylenimine (PEI–PLL) copolymer nanoparticles containing plasmid encoding shRNA targeting anti-apoptotic protein Bcl-xL.94 This conjugate showed much higher specificity for delivering the plasmid encoding Bcl-xL targeted shRNA into nucleolin positive lung cancer A549 cells than that of nanoparticles without aptamer conjugation, and a significant cell apoptosis was observed in nucleolin positive cells but not in nucleolin negative cells (e.g., fibroblast L929), indicating that aptamer-based delivery of therapeutic RNAs is a promising approach for targeted RNA interference cancer therapy.

5. Aptamer based internal radiotherapy

Radioisotopes (e.g., 18F, 123I) are able to dissipate excess energy by spontaneously emitting radiation in the form of alpha, beta, and gamma rays. They are widely applied as probes in cancer diagnosis for the detection and quantification of tumor biomarker expression or PET imaging, and subsequently, radionuclides emitting alpha or beta particles could perform as therapeutic agents because of their tumoricidal effect.95 Iodine-131 (131I), an radioisotope of iodine, is successfully used for the treatment of thyroid carcinoma, because iodine can be specifically accumulated in thyroid tissues to participate in the production of hormones thyroxine and triiodothyronine.96 However, with respect to other radioisotopes such as 90Y and 177Lu, their clinical application for cancer treatment is limited due to the lack of specificity, which results in severe complications.97 Fortunately, targeted delivery systems that specifically deliver radioisotopes into cancer cells expand the application area of radioisotopes and give an opportunity for the improvement of traditional radiotherapy. In 2002, the first targeted radiopharmaceutical, 90Y-ibritumomab-tiuxetan (Zevalin®), was approved by the FDA for the treatment of relapse or refractory low grade, follicular non-Hodgkin's lymphoma (NHL).98

In recent years, several aptamers have also been radiolabeled with radioisotopes such as 99mTc, 18F, 64Cu for tumor imaging (Fig. 3g).99–101 Jacobson et al. labeled a PTK7 targeted aptamer Sgc-8 with 18F via a click chemistry reaction and this conjugation exhibited a higher uptake in PTK7 positive tumors than PTK7 negative tumors, indicating that the radiolabeled aptamer 18F-Tr-Sgc8 could be helpful for the quantification of PTK7 expression in different tissues.100 Li et al. reported a 64Cu labeled aptamer 64Cu-CB-TE2A-AS1411through chelator CB-TE2A for microPET imaging of tumor tissues.101 However, aptamers radiolabeled with therapeutic radioisotopes have not been reported yet. Based on the success of radiolabeled aptamers on tumor targeted imaging, the conjugates of aptamers with therapeutic radioisotopes might also be a promising direction for the development of targeted internal radiotherapy.

6. Aptamer-based combination therapy for overcoming drug resistance

It is worth noting that multidrug resistance (MDR) is one of the most challenging problems for cancer treatment, where cancer cells become very tolerant to anticancer drugs due to genetic mutations, epigenetic changes, upregulation of drug pumps or abnormal activation of signalling pathways.102–104 Clinical data indicated that more than 90% cases of chemotherapy failure are related to the acquirement of MDR. Recently, drug combinations are deemed to be one of most important approaches for overcoming drug resistance, thereby scientists are pioneering many different approaches for delivering multiple drugs into cancer cells as targeted combination therapy.105–107

Among the multiple mechanisms of drug resistance, up-regulation of drug pumps (i.e., ATP-binding cassette transporters, ABC transporters) in cancer cells is most closely associated with drug resistance. These transporters could pump chemotherapy drugs from the inside to the outside of cancer cells, leading to the reduction of intracellular drug levels as well as decrease of therapeutic efficacy.108 P-glycoprotein (P-gp, also known as MDR1) is one of the common ABC transporters that play a major role in conferring resistance to multiple chemotherapies because of its broad substrate specificity to several chemotherapy drugs.108,109 Thus, down-regulating the expression of P-gp could effectively help chemotherapy drugs bypass the capture of ABC drug efflux pumps and circumvent MDR. Several siRNAs have been reported that could markedly inhibit the expression of P-gp, resulting in the restoration of chemotherapy drug sensitivity in MDR cancer cells.110 Wu et al. constructed a KK1B10 aptamer modified nanoparticle containing Dox and MDR-1 targeted antisense oligonucleotides (ASOs), which can specifically deliver the Dox and ASOs into Dox-resistant K562 cells.111 Excitingly, it showed a much stronger anticancer effect on drug resistant cancer cells than that of free Dox treatment, suggesting that the ASOs transported by this nanostructure with the aptamer could efficiently rescue the drug susceptibility.

The impairment of mitochondrial dynamics frequently occurred and are found to be also highly associated with drug resistance in MDR cells; therefore, mitochondria might be a potential therapeutic target to overcome the MDR.112 Notably, B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein and its overexpression on the mitochondrial membrane of cancer cells could protect cells against death.113 Therefore, silencing of the Bcl-2 gene is considered to be an effective strategy for reversing drug resistance. Jeong et al. designed Dox-incorporated multivalent aptamer–siRNA conjugates, in which multimeric antisense siRNA targeting Bcl-2 is annealed with an aptamer-sense siRNA hybrid, and this multivalent conjugate specifically delivered both Dox and Bcl-2 siRNA into drug resistant MCF-7 cells, finally inducing cell death.114 Pan et al. designed and synthesized a Dox contained DNA origami anchored with both Bcl-2 and P-gp ASOs through disulfide bonds. Moreover, twelve additional MUC-1 targeted aptamer sequences are placed on the two sides of the origami for enhancing the targeting ability. This complex could efficiently deliver and controllably release Dox and ASOs into the intracellular space of cancer cells simultaneously, and enhance cancer therapy in adriamycin resistant HeLa and MCF-7 cells, suggesting that simultaneous silencing of P-gp and Bcl-2 genes may also be a great strategy for overcoming the drug resistance.115

7. Automatic design of aptamers through artificial intelligence (AI)

In fact, aptamer selections using existing SELEX approaches are time consuming with low reliability and reproducibility, and the characterization of the aptamer candidates from an enriched oligonucleotide pool with thousands of sequences is also laborious. Thus, more efficient strategy needs to be developed for rapid aptamer screening. Artificial intelligence (AI), which utilizes deep/machine learning algorithms to model intelligent behavior, has been greatly developed for application in almost every field of medicine, including drug development, imagine analysis, patient care, and operational decisions.116,117 Recently, Google's DeepMind team constructed a neural network-based model, AlphaFold, which can predict protein structures with atomic precision, indicating the great potential of AI technology in structural study.118 Moreover, Townshend et al. designed another neural network called the Atomic Rotationally Equivariant Scorer (ARES) for evaluating the root mean square deviation (RMSD) of the provided RNA structure, which is a huge step for RNA structure prediction.119 Along with the study of structural information of RNA and ssDNA, machine/deep learning methods would be developed for the prediction of aptamer structures or the binding affinity of aptamers with targets. Thus, the future direction of aptamer screening is not restricted to the SELEX method, rather than the de novo design of aptamers through the computational method, which we call AI based automatic design of aptamers (i.e., AIptamer) (Fig. 4). In this concept, AI would first analyze the potential binding domain of target proteins, and then design the optimal structure of RNA or ssDNA aptamers that would have the highest binding affinity with their target domain, and finally the sequence of aptamers with the predicted structure is automatically speculated. In this approach, researchers only need to provide information about the target proteins, and AI could provide the output of specific binding aptamers. This concept will dramatically shorten the time and cost for aptamer selection from a huge amount of oligonucleotide library as well as increase the reliability for clinical applications.
image file: d1tb02098f-f4.tif
Fig. 4 Schematic representation of the concept of artificial intelligence (AI) based automatic design of aptamers. First, the potential binding domain of target proteins is analyzed using deep learning/machine learning technologies. Second, based on the potential binding domain, the optimal structure of the ssDNA or RNA aptamers is rapidly predicted by artificial intelligence. Finally, the sequence of aptamers with a predicted structure is automatically speculated, and the binding of the designed aptamer with the target is further validated in the biological system. Created with http://BioRender.com.

8. Conclusions

Targeted therapy (i.e., small molecules or monoclonal antibodies) is a preeminent therapeutic approach to precisely identify and attack certain types of cancer cells, which can also be combined with chemotherapeutic agents, surgery or radiation therapy. Recently, the newly arisen affinity molecule aptamers have acted not only as therapeutic agents but also as effective carriers for targeted delivery of anticancer agents or RNAs into cancer cells to exert their anticancer activity, while keeping damage to healthy cells at a minimum. Thereby, aptamers are promising affinity molecules that can be a replacement for antibodies due to their remarkable chemical properties. Although aptamers have many advantages over antibodies, there are certain challenges that remain. First, clinical applications of aptamer-based drugs are hampered due to their biological characteristics, including rapid clearance in the blood and enzymatic degradation by nucleases. Recent studies have revealed that chemical modifications or substitution of natural bases in the aptamers is able to improve their half-life in vivo. Second, high-throughput selection methods are urgently needed to screen aptamers with clinical potential. Therefore, deeper investigation on the structural basis of the aptamer–target binding complex may help in guiding the future development of aptamer-based precision medicine, while artificial intelligence (AI) including machine/deep-learning methods could be a promising tool for solving the above mentioned problems in near future.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The authors wish to acknowledge the National Natural Science Foundation of China (No. 81872942, 82170143, 82172599), and the China Postdoctoral Science Foundation Funded Project (2020M681901), supported by the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents.

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Footnote

These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2022