Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets (“apatopes”) with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer–aptamer, aptamer–nonaptamer biomacromolecules (siRNAs, proteins) and aptamer–nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe(3)O(4)) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

Aptamer selection and applications for breast cancer diagnostics and therapy

Aptamers are short non-coding, single-stranded oligonucleotides (RNA or DNA) developed through Systematic Evolution of Ligands by Exponential enrichment (SELEX) in vitro. Similar to antibodies, aptamers can bind to specific targets with high affinity, and are considered promising therapeutic agents as they have several advantages over antibodies, including high specificity, stability, and non-immunogenicity. Furthermore, aptamers can be produced at a low cost and easily modified, and are, therefore, called “chemical antibodies”. In the past years, a variety of aptamers specifically bound to both breast cancer biomarkers and cells had been selected. Besides, taking advantage of nanomaterials, there were a number of aptamer-nanomaterial conjugates been developed and widely investigated for diagnostics and targeted therapy of breast cancer. In this short review, we first present a systematical review of various aptamer selection methods. Then, various aptamer-based diagnostic and therapeutic strategies of breast cancer were provided. Finally, the current problems, challenges, and future perspectives in the field were thoroughly discussed.     

Cell-specific aptamer-mediated targeted drug delivery

Nucleic acid aptamers are in vitro-selected small, single-stranded DNA or RNA oligonucleotides that can specifically recognize their target on the basis of their unique 3-dimensional structures. Recent advances in the development of escort aptamers to deliver and enhance the efficacy of other therapeutic agents have drawn enthusiasm in exploiting cell-type-specific aptamers as drug delivery vehicles. This review mainly focuses on the recent developments of aptamer-mediated targeted delivery systems. We also place particular emphasis on aptamers evolved against cell membrane receptors and possibilities for translation to clinical applications.     

Inhibition of cell proliferation by an anti-EGFR aptamer

Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (K_d = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents.     

Organelle-targeted delivery of biological macromolecules using the protein transduction domain: potential applications for Peptide aptamer delivery into the nucleus

Extensive effort is currently being expended on the innovative design and engineering of new molecular carrier systems for the organelle-targeted delivery of biological cargoes (e.g., peptide aptamers or biological proteins) as tools in cell biology and for developing novel therapeutic approaches. Although cell-permeable Tat peptides are useful carriers for delivering biological molecules into the cell, much internalized Tat-fused cargo is trapped within macropinosomes and thus not delivered into organelles. Here, we devised a novel intracellular targeting technique to deliver Tat-fused cargo into the nucleus using an endosome-disruptive peptide (hemagglutinin-2 subunit) and a nuclear localization signal peptide. We show for the first time that Tat-conjugated peptide aptamers can be selectively delivered to the nucleus by using combined hemagglutinin-2 subunit and nuclear localization signal peptides. This nuclear targeting technique resulted in marked enhancement of the cytostatic activity of a Tat-fused p53-derived peptide aptamer against human MDM2 (mouse double minute 2) that inhibits p53-MDM2 binding. Thus, our technique provides a unique methodology for the development of novel therapeutic approaches based on intracellular targeting.     

DNA-aptamer/protein interaction as a cause of apoptosis and arrest of proliferation in Ehrlich ascites adenocarcinoma cells

New prospects for the applications of single-stranded DNA and RNA as therapeutic agents have been discovered in the recent years. Aptamers are the oligonucleotides that bind to their targets with high affinity and specificity due to the well-defined tertiary structures and spatial charge distribution. Aptamers can be selected for any molecules, virus particles, bacteria, cells, and tissues. They have a wide range of applications from target identification to drug delivery. Aptamers themselves can affect various cell functions by affecting certain proteins and receptors. Here, we present the technique for selecting aptamers with antitumor activity in cancer cell cultures and identifying their target proteins by mass spectrometry analysis. The evolved aptamers showed the following antitumor properties: AS-14 (K _d = 3.8 nM) induced apoptosis (phosphatidylserine translocation determined with Annexin V Alexa Fluor 488) and AS-9 (K _d = 0.75 nM) stopped proliferation (as determined with CellTrace? Far Red DDAO-SE) in the culture of Ehrlich ascites adenocarcinoma cells. Using high performance liquid chromatography and high resolution tandem mass spectrometry, we have identified the proteins affected by the AS-14 and AS-9 aptamers. One of the most likely targets for AS-14 was filamin A, which is involved in metastasis formation, tumor development, and cell proliferation. According to mass spectrometry data, the AS-9 aptamer influences the α-subunit of mitochondrial ATP synthase, the key component of mitochondrial oxidative phosphorylation, stimulation of which leads to tumor growth suppression. Thus, mass spectrometry data confirmed the results of the experiments on cell cultures showing that the aptamer binding to specific protein targets causes apoptosis and stops proliferation of cancer cells. However, the mechanisms of action of aptamers in vitro and in vivo are not clear enough and still need to be determined. Our study opens up new possibilities for creation of non-toxic drugs based on DNA aptamers for targeted anticancer therapy.     

综述: 适配子介导的siRNA转运

小分子干扰RNA(small interfering RNA,siRNA)因能快速抑制哺乳动物特定基因的表达而用于各种疾病的治疗,然而选择合适的载体将siRNA安全有效地转运进入靶细胞仍是制约siRNA应用于临床治疗的重要因素．越来越多的转运载体被开发出来,其中包括针对细胞表面蛋白的适配子(aptamer)．Aptamer是一种能与靶分子高特异性和高亲和结合的寡核苷酸,已经越来越多地用于疾病的诊断和治疗．Aptamer作为载体介导siRNA转运可提高治疗的靶向性并减少副作用,这将为siRNA应用于临床靶向治疗提供一种特异有效的途径．目前,已经发现几种aptamers可以介导siRNA的转运,如anti-PSMA aptamer,anti-HIV gp120 aptamer,anti-CD4 aptamer等．本文将综述aptamer介导siRNA转运的最新研究进展．     

Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer

Cell penetrating peptides (CPPs) are short amphipathic and cationic peptides that are rapidly internalized across cell membranes. They can be used to deliver molecular cargo, such as imaging agents (fluorescent dyes and quantum dots), drugs, liposomes, peptide/protein, oligonucleotide/DNA/RNA, nanoparticles and bacteriophage into cells. The utilized CPP, attached cargo, concentration and cell type, all significantly affect the mechanism of internalization. The mechanism of cellular uptake and subsequent processing still remains controversial. It is now clear that CPP can mediate intracellular delivery via both endocytic and non-endocytic pathways. In addition, the orientation of the peptide and cargo and the type of linkage are likely important. In gene therapy, the designed cationic peptides must be able to 1) tightly condense DNA into small, compact particles; 2) target the condensate to specific cell surface receptors; 3) induce endosomal escape; and 4) target the DNA cargo to the nucleus for gene expression. The other studies have demonstrated that these small peptides can be conjugated to tumor homing peptides in order to achieve tumor-targeted delivery in vivo. On the other hand, one of the major aims in molecular cancer research is the development of new therapeutic strategies and compounds that target directly the genetic and biochemical agents of malignant transformation. For example, cell penetrating peptide aptamers might disrupt protein-protein interactions crucial for cancer cell growth or survival. In this review, we discuss potential functions of CPPs especially for drug and gene delivery in cancer and indicate their powerful promise for clinical efficacy.     

Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection

BackgroundThe broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.

Methodology/Principal Findings

We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.

Conclusions and Significance

We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.     

Application of aptamers in diagnostics,drug-delivery and imaging

Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their target with high specificity and affinity. Although aptamers are analogous to antibodies for a wide range of target recognition and variety of applications, they have significant advantages over antibodies. Since aptamers have recently emerged as a class of biomolecules with an application in a wide array of fields, we need to summarize the latest developments herein. In this review we will discuss about the latest developments in using aptamers in diagnostics, drug delivery and imaging. We begin with diagnostics, discussing the application of aptamers for the detection of infective agents itself, antigens/toxins (bacteria), biomarkers (cancer), or a combination. The ease of conjugation and labelling of aptamers makes them a potential tool for diagnostics. Also, due to the reduced off-target effects of aptamers, their use as a potential drug delivery tool is emerging rapidly. Hence, we discuss their use in targeted delivery in conjugation with siRNAs, nanoparticles, liposomes, drugs and antibodies. Finally, we discuss about the conjugation strategies applicable for RNA and DNA aptamers for imaging. Their stability and self-assembly after heating makes them superior over protein-based binding molecules in terms of labelling and conjugation strategies.     

Development of HBsAg-binding aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen(HBsAg),a specific antigen on the membrane of Hepatitis B virus (HBV)-infected cells,provides a perfect target for therapeutic drugs.The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection.Herein,we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes.One high affinity aptamer,HBs-A22,was isolated from an initial 115 mer library of ~1.1×10 15 random-sequence RNA molecules using the SELEX procedure.The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells.This is the first reported RNA aptamer which could bind to a HBV specific antigen.This newly isolated aptamer could be modified to deliver imaging,diagnostic,and therapeutic agents targeted at HBV-infected cells.     

In vitro selection of cell-internalizing 2′-modified RNA aptamers against <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Infectious diseases caused by bacterial or viral agents represent the major cause of human pathogenesis and mortality worldwide. A development of novel antibacterial therapeutics and diagnostic tools is a very acute task. The use of DNA and RNA aptamers targeted to certain bacteria could be a promising solution to this problem. Here, we propose a new protocol of selection of 2′-fluoro RNA aptamers capable to internalize into bacterial cells. Using whole-cell SELEX against Pseudomonas aeruginosa, enriched 2′-fluoro RNA library was obtained, and its sequencing and data analysis were fulfilled. It was found that the central region of predominating aptamer sequence is identical to the fragment of P. aeruginosa rRNA. A possibility of internalizing of this aptamer into bacterial cells is shown. It is hypothesized that aptamers could be internalized more effectively as heterodimeric complexes.     

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K_d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.     

Aptamers: Molecules of great potential

Aptamers emerged over 20 years ago as a class of nucleic acids able to recognize specific targets. Today, aptamer-related studies constitute a large and important field of biotechnology. Functional oligonucleotides have proved to be a versatile tool in biomedical research due to the ease of synthesis, a wide range of potentially recognized molecular targets and the simplicity of selection. Similarly to antibodies, aptamers can be used to detect or isolate specific molecules, as well as to act as targeting and therapeutic agents. In this review we present different approaches to aptamer application in nanobiotechnology, diagnostics and medicine.     

Novel aptamer-nanoparticle bioconjugates enhances delivery of anticancer drug to MUC1-positive cancer cells in vitro

MUC1 protein is an attractive target for anticancer drug delivery owing to its overexpression in most adenocarcinomas. In this study, a reported MUC1 protein aptamer is exploited as the targeting agent of a nanoparticle-based drug delivery system. Paclitaxel (PTX) loaded poly (lactic-co-glycolic-acid) (PLGA) nanoparticles were formulated by an emulsion/evaporation method, and MUC1 aptamers (Apt) were conjugated to the particle surface through a DNA spacer. The aptamer conjugated nanoparticles (Apt-NPs) are about 225.3 nm in size with a stable in vitro drug release profile. Using MCF-7 breast cancer cell as a MUC1-overexpressing model, the MUC1 aptamer increased the uptake of nanoparticles into the target cells as measured by flow cytometry. Moreover, the PTX loaded Apt-NPs enhanced in vitro drug delivery and cytotoxicity to MUC1(+) cancer cells, as compared with non-targeted nanoparticles that lack the MUC1 aptamer (P<0.01). The behavior of this novel aptamer-nanoparticle bioconjugates suggests that MUC1 aptamers may have application potential in targeted drug delivery towards MUC1-overexpressing tumors.     

Targeted delivery of chemotherapy agents using a liver cancer-specific aptamer

Background

Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.

Methodology/Principal Findings

In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.

Conclusions/Significance

Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general.     

前列腺癌适配体的研究进展及其应用

寻找一种高特异性高灵敏度的方法对于前列腺癌的早期发现和及早干预,进而提高患者的生存率及生活质量十分重要。适配体(Aptamer)是由一段寡聚核苷酸链折叠形成的特定三维结构,能高亲和性、高特异性地靶向不同的分子。自上世纪90年代,许多研究者致力于适配体的研究,目前,适配体在药物递送、肿瘤诊疗等方面的研究已取得较大进展。本文将对前列腺癌适配体新近的研究进展及其应用进行综述,以期为将来进一步的研究和临床应用提供参考。     

Induction of splice correction by cell-penetrating peptide nucleic acids

BACKGROUND: Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as beta-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing. METHODS: HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 degrees C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs. RESULTS: All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 degrees C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis. CONCLUSIONS: These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.