Spiegelmer Aptamers: Innovative Approach in Breast Cancer.

1. Introduction

Breast cancer is classified by the expression of estrogen and progesterone receptors (ER and PR), as well as human epidermal growth factor receptor 2 (HER2) status, whereas triple-negative breast cancer (TNBC) lacks all of these markers. Significant advances have been made in conventional breast cancer treatments, including chemotherapy, hormone therapy, immunotherapy, radiotherapy, and surgery. Ongoing research is focused on overcoming drug resistance, preventing recurrence and metastasis, and developing combination therapies to improve patient survival and quality of life. Nevertheless, the number of deaths due to breast cancer was 685,000 in 2020 and decreased to 670,000 in 2022, highlighting the ongoing need for enhanced research and the development of more effective methods for early diagnosis and treatment. 1 - 3 Immunohistochemistry (IHC) and MammaPrint technique, which utilizes gene expression analysis, provides valuable information about tumor characteristics, serving as biomarkers for guiding targeted therapy, treatment response, and assessing the risk of recurrence and metastasis. 4 , 5 The discovery and development of new biomarkers for breast cancer, particularly TNBC, are crucial for advancing treatment approaches such as personalized medicine, photodynamic therapy (PDT), targeted therapies, and early diagnosis. 3 Cancer biomarkers can be targeted by antibodies, peptides, aptamers and small molecules for therapeutic and diagnostic applications. Unlike small molecules, which do not bind to biomarkers specifically, aptamers not only exhibit high specificity in biomarker binding but are also small in size and exhibit low immunogenicity and stability compared to antibodies and peptides. Additionally, aptamers possess the unique ability to bind to and inhibit miRNA biomarkers, which makes them superior for targeting such biomarkers. These advantages suggest that aptamers hold greater potential as a focus for future research compared to other molecular groups. 6 , 7

Nearly 35 years have passed since the discovery of the first aptamer. During this time, two aptamers have received food and drug administration (FDA) approval for therapeutic use, while many others are currently undergoing various phases of clinical trials. One major challenge hindering the widespread adoption of aptamers as therapeutic agents is their nucleotide-based structure, which makes them susceptible to degradation by nucleases in biological systems. 8 Extensive research has focused on modifying aptamer structures to enhance their stability in bodily fluids, resulting in the development of novel aptamer variants. Notably, spiegelmers—a unique class of aptamers composed of mirror-image sugars (L-nucleotides) in their backbone—have emerged. This distinctive architecture confers resistance to nucleases, preventing their degradation in biological systems. Spiegelmers fold into a three-dimensional structure composed of 30 to 50 L-nucleotides, enabling them to specifically target a range of substances, including amino acids like arginine, molecules such as adenosine, peptides like hepcidin, as well as proteins and RNA. 9 Consequently, the development of spiegelmers provides a promising foundation for enhancing biomarker-targeted therapies. In addition to therapeutic applications, spiegelmers can also be used in diagnostics; for example, spiegelmers conjugated to radioactive isotopes or fluorescent markers can be employed in positron emission tomography (PET) and magnetic resonance imaging (MRI). 10 , 11 Additionally, owing to their remarkable stability, spiegelmers offer a promising new approach for real-time imaging during lumpectomy surgery, enabling precise determination of tumor margins. Taken together spiegelmers represent promising candidates for theranostic development. In this review, we discuss spiegelmers as a class of aptamers with exceptional properties, which lays the basis for future research into their therapeutic and diagnostic applications in breast cancer.

2. Definition and Characteristics of Aptamers

Aptamers are classified into two categories based on their structural framework: peptide aptamers and nucleic acid aptamers. Peptide aptamers bind to target molecules via a peptide loop composed of 8 to 20 amino acids. This peptide loop is stabilized and specifically configured by a scaffold protein. Peptide aptamers can bind to intracellular targets and modulate their functions, enabling therapeutic applications in cancer and antiviral treatments, as well as use in diagnostic tools like protein microarrays. While Nucleic acid aptamers are single-stranded oligonucleotides, including DNA, RNA, xeno nucleic acids (XNA) and circular aptamers, typically consisting of 25–80 nucleotides and having a molecular weight of less than 20 kDa. 12 - 14 Each type of Nucleic acid aptamers offers distinct advantages: RNA aptamers, due to their smaller size, can penetrate cells more easily and offer greater flexibility in binding to their targets, whereas DNA aptamers demonstrate enhanced stability because of their deoxyribose sugar backbone. However, the stability of these simple oligonucleotides is limited-typically less than an hour inside living cells and around five minutes in serum. Consequently, researchers are actively developing a variety of aptamers to overcome these limitations. 8 , 15 Modifying the nucleic acid sugar group, such as adding a methyl group to the 2′-hydroxyl of ribose (2′-OMe), produces XNAs that are more stable and exhibit enhanced functional properties. 12 Joining the 3′-terminus of an exon to the 5′-terminus of the same or an upstream exon via chemical or ligase methods produces circular aptamers that are resistant to exonucleases, resulting in enhanced stability. This circularization also increases the thermal stability of the aptamer and minimizes nucleotide modifications. Another modification they implemented is linking two circular aptamers known as circular bivalent aptamers (cb-aptamers) which significantly improves binding affinity. 16 , 17

In addition strategies to extend the half-life of aptamers have also been developed.Aptamers exhibit high renal filtration because of their low molecular weight. One of the most common strategies to reduce renal clearance is PEGylation of the aptamer; however, this modification can introduce spatial constraints. Alternative approaches include attaching cholesterol, proteins such as albumin, or nanomaterials to the 5′ end of the aptamer to enhance its stability and reduce filtration. 13 The three-dimensional structure formed by this single-stranded sequence enables the aptamer to bind a wide range of targets including cells, viruses, bacteria, organic molecules, proteins, sugars, metal ions, transcription factors, microRNAs, siRNAs, and cofactors through hydrogen bonding, van der Waals forces, π-π stacking, and electrostatic interactions. 18 - 21

The aptamer that specifically binds to the target molecule is identified in vitro using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). In this process, the target molecule is exposed to a library of randomized aptamers, and those that bind are isolated and enriched through several selection cycles. The final aptamer is then sequenced, and additional modifications can be made to optimize its properties. 22 Also To enhance the efficiency of aptamer design, in silico methods are employed, including tertiary structure modeling, docking to identify complexes with the lowest binding energy, and molecular dynamics simulations. 23 In recent years, numerous studies have focused on developing aptamer synthesis methods. Additionally, machine learning techniques such as two-layer neural networks and Restricted Boltzmann Machines (RBMs) have been employed to predict aptamer-target binding, thereby shortening the SELEX process. 24

3. Applications of Aptamers Derived From Their Unique Properties

To date, numerous aptamers have been identified with a wide range of potential applications. Although aptamers are often called chemical antibodies, they demonstrate greater stability across a wide range of temperatures and pH levels compared to conventional antibodies, with the added advantage of reversible thermal denaturation,chemically synthesized through a faster and more cost-effective process, exhibit no batch-to-batch variation, have a longer half-life, are easier to manipulate, and possess low immunogenicity.Consequently, aptamers represent a promising alternative to antibodies for therapeutic and diagnostic applications. 8 , 21 , 22

Aptamers have high affinity for target molecules even at low concentrations which make aptamers excellent candidates for diagnostic use. Moreover, their ability to be conjugated with fluorescent dyes and radioisotopes further enhances their utility in imaging and detection techniques. 22 , 25 , 26 For instance aptamer MF3Ec, a DNA aptamer used in aptahistochemistry assays, enables the discrimination of normal breast tissue from Luminal A breast cancer tissue, para-carcinoma tissue, and mastitis tissue. 27 A circular aptamer designed for leukemia targets two different markers on CCRF-CEM cells. This dual aptamer combines the XQ-2d and Sgc8c sequences, enhancing the potential for more precise cancer detection and advanced diagnostic applications. 28 Additionally, aptamers can be used in flow cytometry for immunophenotyping blood cells and in ELISA assays. 22

Among bioreceptors that recognize analytes, aptamers present a promising alternative to antibodies, as they enhance both the sensitivity and selectivity of biosensors factors crucial for the early detection of cancer biomarkers. Currently, electrochemical, optical, and mass-sensitive aptasensors are being actively developed. 25 , 29

Furthermore, aptamers, with their unique characteristics, can serve as an alternative to antibodies in therapy. For example, Dario Ruiz-Ciancio and colleagues developed an aptamer targeting the CD22 protein to treat B cell acute lymphoblastic leukemia (B-ALL), offering a possible alternative to antibody-drug conjugates (ADCs) and chimeric antigen receptor T (CAR-T) therapies. 30 Aptamers exhibit strong penetration into tumor tissues. Additionally, circular aptamers can specifically target tumor cells circulating in the bloodstream, making them a promising option for cancer treatment. 16 , 17 The development of aptamers holds significant potential for clinical applications beyond in vitro and animal studies, as evidenced by two aptamers that have already received FDA approval for clinical use. Pegaptanib is the first FDA-approved RNA aptamer featuring nucleotide modifications that enhance its stability against nucleases. This aptamer binds with high affinity to its target, VEGF165, effectively inhibiting it, and has been primarily used to treat age-related macular degeneration (AMD). However, its applications are expanding to other diseases. Another FDA-approved aptamer, pegylated avacincaptad pegol, acts as an inhibitor of complement factor 5 (C5) and is used in the treatment of geographic atrophy (GA) secondary to AMD. 8 Aptamers can be utilized to deliver therapeutic agents specifically to cancerous and diseased tissues. These include peptide-conjugated aptamers, nucleic acid-conjugated aptamers, and drug-conjugated aptamers. The conjugation of aptamers to drugs can occur via nucleic acid synthesis, covalent bonding, or physical interactions. Additionally, aptamers can be linked to liposomes for drug delivery or combined with various nanocarriers, including both organic and inorganic nanomaterials. 21 , 26 , 31 , 32

4. The Concept of Spiegelmers

As mentioned, chemical modifications, such as incorporating 2′-amino nucleotides, were used to increase the stability of aptamers. However, these modifications can affect the specificity of aptamer binding to the target molecule. Despite these improvements, aptamers still exhibit some instability in biological environments. To address this, a new generation of aptamers called spiegelmers was developed; due to their unique structure, they are resistant to DNases and RNases degradation in body fluids. Spiegelmers, also known as biostable or mirror-image aptamers, contain L-(deoxy) ribose instead of the natural D-nucleotides. Unlike the right-handed helix found in traditional aptamers, spiegelmers form a left-handed helix ( Figure 1 ). 9 , 33 Spiegelmers can be composed of L-DNA or L-RNA. Although their building blocks are unnatural, they bind to their targets in a natural configuration. 34 , 35

The first L-RNA spiegelmers, developed in 1996 for the small molecules arginine and adenosine, demonstrated remarkable resistance to nucleases, resulting in their stability in human serum for over 60 hours. This breakthrough led to the expanded study and application of spiegelmers. In 1997, the first biologically active L-DNA spiegelmer was introduced, targeting the peptide hormone vasopressin. Spiegelmers are similar to other aptamers, typically consisting of 30-50 nucleotides that fold into a three-dimensional structure and selectively bind various molecules. 9 , 36 Some examples of spiegelmers include: L-aptamer binding to D-TAR RNA is related to the HIV virus, which inhibits the formation of a complex with the Tat protein 37 .The 40-nucleotide L-aptamer, NOX-D20, which consists of a combination of L-RNA and L-DNA, binds to the C5a inflammatory mediator. 38 Inhibition of calcitonin gene-related peptide by antibody is effective in relieving migraine headaches. L- Aptamer, NOX-C89, can bind to this peptide and be effective in relieving trigeminal spinal activity. 39 These were examples of therapeutic applications of spiegelmer that bind to RNA, protein, and peptide targets ( Figure 2 ).

Notably, spiegelmers do not form Watson-Crick (WC) base pairs with natural D -nucleotides, so they can be bind through their three-dimensional shape, Thereby they bind to the target with high affinity and specificity. For example, a spiegelmer made against the peptide hormone, gonadotropin-releasing hormone (GnRH), binds to it with high affinity (K D = 20 nM). 40

So spiegelmer has fewer off-target effects similar to aptamers and it can considerable as diagnostic applications. An interesting application is the use of a fluorogenic spiegelmer as a biocompatible sensor that produces fluorescence in the presence of microRNA-155, enabling the identification of microRNAs in living cells. 10

Also, spiegelmer has been used instead of antibodies in the sandwich assay. This study showed that spiegelmer binding to the C-terminal region of cardiac troponin I, a biomarker of acute coronary syndrome, is suitable for protein-specific detection. 41

In addition to the properties mentioned above, the permeability of spiegelmers makes them well-suited candidates for imaging modalities such as PET. Investigation of the distribution of labeled L-RNAs and L-DNAs in vivo using the PET method showed that spiegelmers are suitable for imaging applications due to their good metabolic stability and pharmacological properties. 42

Furthermore, mirror-image aptamers are useful in the separation and purification of molecules by chromatography, such as chiral stationary phase. 43

Therefore, spiegelmers represent a promising alternative to antisense oligonucleotides and antibodies for both therapeutic and diagnostic applications. Table 1 presents spiegelmers and their corresponding applications.

Like aptamers, spiegelmers have high renal clearance and short half-life due to their low molecular weight, and one way to increase their stability is PEGylation. PEG is FDA approved for pharmaceuticals use. The RNA spiegelmer, NOX-H94, plays a role in inhibiting hepcidin and causing an increase in blood iron. PEGylation of this spiegelmer increased its half-life in serum to 14 to 26 hours. 13

Similar to aptamers, SELEX are used to synthesize spiegelmers, but natural RNA polymerase cannot use L-nucleotides to synthesize oligonucleotide strands. Therefore, the target molecule must be converted to the enantiomer form and a library of 10 15 or more D -oligonucleotides is used to isolate and amplify the aptamer binding to the enantiomer of the target molecule during cycles. Ultimately, the selected aptamer serves as a template for spiegelmer synthesis, and the L form of the oligonucleotide can bind to the natural form of the target molecule. Reversed-phase HPLC is used for purification, and tangential flow ultrafiltration is used for desalting. Proper purification of spiegelmer is essential for proper folding and binding to its target. 56 , 57

5. Spiegelmers: An Alternative to Monoclonal Antibodies

Both spiegelmers and monoclonal antibodies (mAbs) have high affinity for binding to their ligands. mAbs bind with high specificity to a single epitope of their target antigen, and the targeted binding of mAbs has led to the expansion of studies and production of mAbs and has made them a powerful tool in the field of numerous diseases, including cancer, blood diseases, inflammatory diseases, infectious diseases, and autoimmune diseases, so far mAbs are one of the best-selling categories of biological molecules on the market, and even during the COVID-19 epidemic, studies were conducted on the development of antibodies to treat it. However, spiegelmers also bind to natural target with high affinity due to their reciprocal chirality, but they are not as popular as mAbs. 40 , 58 , 59

The shelf life of spiegelmers is 3 years when stored at 2-4 °C and is lyophilized, but antibodies have a limited shelf life when stored at 2-4°C and is sensitive to temperature changes.Antibodies are degradate by proteases in the body, but spiegelmers are resistant to proteases and nucleases. 9 , 21 mAbs, even fully human mAbs, can induce the immune system and make an antidrug antibodies (ADA), which is accompanied by antibody clearance and decrease its effectiveness. Also, the immune response stimulation can affect the effectiveness of drugs bound to mAbs, while so far the use of spiegelmers has not been associated with an immune response or toxicity. 60 , 61 A significant advantage of spiegelmer is having small targets that, due to their size, are unable to elicit an immune response to the substances, and mAbs therapies cannot be used for this purpose. 21 , 62

The stability of mAbs is due to the presence of the constant fragment (Fc) domain of a human immunoglobulin (Ig) G and its role in recycling following binding to the neonatal Fc receptor (FcRn). Studies have shown that conjugation of the Fc domain to drugs, including factor IX-Fc (Alprolix), plays a role in increasing its half-life. 13 , 63 The attachment of this domain to spiegelmer could be considered to increase its half-life and also reduce the renal clearance of spiegelmer.

The initial technology for producing mAbs was the hybridoma technique, which limited the amount of antibody, but despite the challenges in its production, many advances have been made in the production of mAbs over the years, including the production of chimeric antibodies and transgenic humanized animals that produce high-quality humanized antibodies. Given the significant advantages that spiegelmers have, their synthesis is accompanied by challenges, including the creation of enantiomers of the target molecule and the use of polymerases that bind unnatural nucleotides. 9 , 59 Perhaps in the future, many of these challenges will be resolved through expanded studies.

In addition to the challenge we face in the synthesis of spiegelmer, another obstacle to the expansion of spiegelmer in global markets is the lack of predictability of the spiegelmers behavior in biological systems due to their unnatural framwork. While antibodies are glycoproteins that are also produced in the body against pathogens and have a known behavior in biological systems, and catabolic enzymes that are present everywhere in the body break down antibodies into their amino acid components. 59 , 64 However, mAbs production technology is based on genetically modified cells, and several factors, including commercial-scale mAbs production,temperature, pH and etc., can affect the quality and glycosylation pattern of the antibody and cause differences in batch to batches, while spiegelmers are chemically synthesized and can have a constant production. 54 , 65 Table 2 is a comparison between aptamers, spiegelmer and mAbs.

6. Overview of Breast Cancer and Common Treatments

Breast cancer is a common and increasingly prevalent malignancy among women worldwide. While the 5-year survival rate ranges from 80%-90% in most countries, it remains significantly lower in South and Southeast Asian nations. 67 Breast cancer is diagnosed using Mammography (a key screening method), MRI (which offers greater sensitivity) and breast ultrasound. 68 , 69 This cancer is classified into stages I to IV based on tumor size and extent of spread. However, based on IHC results, tumors that are positive for ER and PR are classified as luminal A, which accounts for approximately 60% of cases and is associated with a favorable prognosis. 70 , 71 This subtype is typically treated with tamoxifen and aromatase inhibitors. 72 However, these treatments are associated with side effects, such as cardiovascular complications, and may lead to disease relapse. 73 , 74 If IHC results are positive for HER2, breast cancer is classified as the HER2 subtype, which accounts for approximately 10–15% of cases and is associated with a poor prognosis and reduced survival. Targeted anti-HER2 mAbs, such as trastuzumab and pertuzumab, are available treatments for this subtype, though both carry potential cardiac side effects. 70 , 75 In addition, the development of resistance limits efficacy, and treatment with anti-HER2 antibodies is beneficial in only approximately one-third of patients. 70 , 76 If ER, PR, and HER2 are positive, the subtype is classified as luminal B, which accounts for approximately 30% of cases and is associated with a poorer prognosis due to high Ki-67 expression, a marker of cell proliferation. 70 , 77

When IHC results are negative for all receptors, the tumor is classified as TNBC. This subtype accounts for 15-20% of cases and often recurs within 3 years after diagnosis. 70 , 78

The lack of biomarkers in TNBC poses significant challenges for the development of targeted therapies consequently, contributes to poor survival outcomes and increased likelihood of metastasis. 79

Following a breast cancer diagnosis, the primary treatment option is surgery, which may involve a mastectomy or lumpectomy. 80 Another treatment option is radiotherapy, which patients may receive either before or after surgery. However, radiotherapy is a lengthy and costly process that can cause adverse side effects. 76 , 81 Chemotherapy is a common treatment for breast cancer, used to shrink tumors prior to surgery, target metastatic disease, provide post-operative therapy, and serve as the primary treatment for advanced cases. Each chemotherapy drug has its own unique side-effects profile. For example, doxorubicin can cause serious heart failure. 71 , 76 Taken together, current cancer treatments are associated with significant side-effects that impair patients’ quality of life, as well as with risks of disease recurrence and metastasis. Therefore, developing novel diagnostic methods and advancing targeted therapies are essential priorities for future research.

7. Advancements in Breast Cancer Treatments

New treatments being developed for breast cancer include PDT, photothermal therapy (PTT), and immunotherapy. PDT utilizes light of a specific wavelength to generate reactive molecules that damage cellular biomolecules and induce tumor cell necrosis, apoptosis. 82 PDT damaging the tumor blood vessels and releasing cytokines that activate the immune system. PDT has demonstrated promising results and minimal side effects. However, its use is currently limited to small tumors. 83 Another emerging therapeutic approach is PTT. In this method, near-infrared (NIR) light irradiation activates photothermal agents, typically nanoparticles, to induce apoptosis in cancer cells. Promising results with and minimal side effects have been observed in breast cancer treatment, so PTT could become a viable option for TNBC and metastatic breast cancer. Furthermore, PTT can be combined with chemotherapy and PDT to achieve synergistic therapeutic effects. 84 , 85

Immunotherapy for breast cancer targeting programmed death-1 (PD-1) and its ligand PD-L1. The effectiveness of mAbs used in immunotherapy varies by breast cancer subtype, with the most favorable response observed in first-line treatment of metastatic TNBC that is PD-L1 positive. This approach, combining pembrolizumab with paclitaxel, nab-paclitaxel, or gemcitabine plus carboplatin, has received FDA approval. 86

Three newly FDA-approved ADC drugs have been developed to treat breast cancer with the aim of reducing drug toxicity. These ADCs are created by linking antibodies, targeting specific cancer cell surface antigens,to cytotoxic drugs via a chemical linker. Among these, trastuzumab duocarmazine and trastuzumab emtansine are two novel therapies for patients with HER2-positive breast cancer. Additionally, sacituzumab govitecan (SG), which combines an antibody targeting trophoblast cell surface antigen 2 with the active metabolite of irinotecan, represents a new treatment option for TNBC. 87

Overall, the heterogeneous nature of breast cancer has driven the development of theranostic approaches that integrate diagnostic imaging agents with therapeutic drugs. 88 To date, numerous molecular targets for breast cancer theranostics have been identified, including HER2, PSMA, SSTR, CXCR4, and FAP. Theranostics have demonstrated promising clinical outcomes. 89 For example, one study utilized trastuzumab conjugated to the bifunctional chelator DOTA, labeled with lutetium-177 (Lu-177), for SPECT/CT imaging to specifically identify HER2-positive patients. 90

Therefore, further research is needed to develop new, cost-effective methods for early detection and treatment that enhance patients’ quality of life by focusing on personalized medicine and identifying key biomarkers in breast cancer. These biomarkers could serve as therapeutic targets for developing mAbs, aptamers, and peptides. Among these novel therapies, spiegelmers offer significant advantages such as stability, high affinity, and the ability to target microRNAs as biomarkers that have been largely overlooked in current targeted therapies. This therapeutic approach holds great promise for the future of breast cancer treatment.

8. Spiegelmer Aptamers in Breast Cancer Diagnosis

Late diagnosis of breast cancer is associated with metastasis, which leads to poor patient survival. Therefore, diagnosis in the early stages of breast cancer is directly related to patient survival and successful treatment. In addition, the expansion of imaging methods used to detect tumor, location and size, the identification of biomarkers is accompanied by the expansion of molecular diagnostic methods that can be useful in early diagnosis and targeted therapy. One advancement in breast cancer detection by MRI is the use of the AS1411 aptamer conjugated to a superparamagnetic iron oxide nanoparticle. This aptamer binds to nucleolin expressed in breast cancer cells, and its development could enhance specificity and sensitivity for tumor detection by MRI. 91 Another example, the use of a HER2 -specific aptamer labeled with 18 F- fluoride has shown promising results in the diagnosis of HER2 -positive breast cancer by PET. 92 Both RNA and DNA spiegelmers enable specific target binding and can be labeled with radiotracers as molecular probes. For example, spiegelmers labeled with 85 Y and 67 Ga for PET imaging have demonstrated high uptake in target areas. 42 Importantly, similar to aptamers, spiegelmers are excellent candidates for tumor imaging due to labeling the spiegelmer does not affect its function or binding affinity.

Fluorescent probes are commonly used for real-time visualization of cellular and molecular processes. 93 Spiegelmers have been developed for diagnostic by fluorescent probes application. Gui-Mei Han and colleagues investigated the stability and efficacy of fluorescent probes that are mirror images of DNA aptamers. Their study showed that the L-DNA version of the AS1411 aptamer, conjugated to silver nanoclusters (Ag NCs) and targeting nucleolin, exhibited enhanced uptake in HeLa cells. 94 Evidence suggests that spiegelmer, with its prominent characteristic of stability in biological systems, is a promising tool for real-time imaging during lumpectomy surgery,which can provide precise image of the tumor margins.

Taken together, spiegelmers,as a generation of aptamer, possess remarkable potential for molecular imaging in breast cancer diagnosis using MRI, PET, and bioimaging.The advancement of spiegelmers in diagnostic applications can facilitate the development of more accurate methods.

Cancer blood markers such as exosomes released from cancer cells, tumor genomic DNA fragments, Circulating tumor DNA (ctDNA), circular microRNAs (circRNAs), and long noncoding RNAs (lncRNA) and circulating tumor cells (CTC) can be suitable options for monitoring tumor. 95 For instance, the examination and counting of CTCs in breast cancer is associated with the response to treatment. Also, the presence of CTCs is predictor of metastasis and recurrence in breast cancer. In addition, in TNBC and HER2 positive breast cancer, the presence of mRNA Cytokeratin 19 (Ck19) in CTCs indicates resistance to treatment and aggressiveness of the breast cancer. 96 In recent years, many studies have been conducted to develop methods for identifying these markers. Although the use of these cancer blood markers is useful in non-invasive and early stages diagnosis of cancer, the main challenge is the very low concentration of these markers in the blood. The development of microfluidics devices could be useful in the detection of low-volume samples such as microRNAs and exosomes. 97 , 98 The separation of markers in a small sample volume is due to the special geometry of the microfluidics devices. The use of oligonucleotides can be an alternative to antibodies in microfluidics devices, so that DNA aptamers immobilized by biotin on the surface of the microfluidics device were able to separate 10 tumor cells in a volume of one ml from peripheral blood with an efficiency of 95% in less than 30 minutes. 99 - 101 Also, RNA aptamer binding to the extracellular domain of ErbB2 could be a suitable option for identifying ErbB2 positive breast cancer cells in the circulation and following treatment. 102 SO, spiegelmers, with their biostability, could be an option for future studies in the field of identifying cancer blood markers by microfluidics devices. Also the advantage of spiegelmers over antibodies is their ability to detect cancer related RNA molecules in the blood.

Gene profiling in breast cancer can be used in targeted therapy, patient survival, metastasis risk, and identification of treatment resistance mechanisms. Microarray analyses are used to identify differentially expressed genes, and comparative genomic hybridization (CGH) is used to analyze chromosomal alterations in breast cancer. 103 MammaPrint was the first genomic assay to identify the expression of 70 genes in breast cancer for targeted therapy. 5 The binding of spiegelmers to their target is in a cross-chiral state, which is based on the sequence and structure of the target nucleotide, creating a third type of interaction, and WC base pairs do not play a role in this binding, which results in highly specific binding and minimal of-target hybridization. 104 This makes L-DNA an excellent candidate for microarrays with enhanced specificity and reduced noise. Also an attractive application of spiegelmers in microarray techniques is to identify DNA-interacting proteins or to identify polymorphisms.Spiegelmers have significant applications in diagnostics through their role in the design of biosensors. These optosensors have the ability to detect the concentration of analytes such as metal ions in living cells. 105 Endocrine-disrupting chemicals such as bisphenol A (BPA), diethylstilbestrol, dioxins and dichlorodiphenyltrichloroethane have been implicated in the development of breast cancer. 106 The development of biosensors to detect these carcinogenic compounds in the blood could provide a warning to exposed individuals and susceptible to breast cancer. In a new approach, spiegelmer-based optosensors were used to detect blood BPA, which had high sensitivity and stability for detection. 107

Hormonal levels are a risk factor for breast cancer, so that in post-menopausal women, high levels of testosterone, insulin-like growth factor-1 (IGF-1), and sex hormone-binding globulin (SHBG) hormones are associated with breast cancer. 108 Therefore, monitoring the levels of these hormones in high-risk individuals could serve as a non-invasive method for cancer prognosis. Spiegelmer-based biosensors can be used to assess hormone levels. Thiolated spiegelmer attached to Au nano particles and molybdenum disulfide nanoflowers/graphene nanoribbons in the form of a nanocomposite has been used in a biosensor platform that reproducibly and stably determines the level of hepcidin, a peptide hormone, in human serum samples with high sensitivity. 109 Therefore,the design and synthesis of spiegelmers that can identify high levels of hormones have the potential to predict the risk of breast cancer. Taken together spiegelmers show strong potential as candidates for diagnostic applications ( Figure 3 ).

9. Advantages of Spiegelmers in Breast Cancer Treatment

Complications and resistance to existing drugs, the heterogeneity of cancer, the absence of effective treatments for TNBC, and the need to improve patients’ quality of life all necessitate the development of new therapeutic approaches for breast cancer. The identification and targeting of biomarkers is linked to enhanced treatment response, reduced drug resistance, and the avoidance of ineffective interventions. Biomarkers can be found in body fluids or tumor tissue. More importantly, they can be used indicators of treatment efficacy, as well as having direct therapeutic applications. 4

microRNAs biomarkers have been less explored as therapeutic targets. miR-21 is a potential target for luminal A subtype, while in luminal B, both miR-21 and miR-145 may be targeted. In HER2-positive breast cancer, which often exhibits resistance to mAbs therapy, miR-125a, miR-125b, and miR-148a are promising therapeutic targets. Furthermore identifying microRNA biomarkers can provide novel therapeutic opportunities for TNBC. 96 Many of the identified biomarkers could have theranostic applications. 110 , 111

A key distinguishing feature of spiegelmers, as aptamers, compared to antibodies is their ability to specifically bind microRNA biomarkers. ‘’aptamiRs’’ are spiegelmers that bind to the stem loop of microRNAs with their three-dimensional structure and prevent the function of Dicer and the formation of mature microRNAs. The advantage of aptamiRs compared to antisense oligonucleotides is the lack of WC base pairing, which leads to high specificity in binding.For example, an L-RNA aptamer with a dissociation constant (Kd) of 19 nM binds to pre-microRNA-155, inhibiting Dicer’s processing and thereby preventing the generation of mature microRNA. Notably, miR-155 is highly expressed in many malignancies. 112 Also, this microRNA is highly expressed in breast cancer and is closely linked to tumor progression and drug resistance. Inhibition of miR-155 has been shown to suppress breast cancer cell growth both in vitro and in vivo, while also enhancing sensitivity to chemotherapy. 113 Evidence indicates that targeting miR-155 with spiegelmer holds significant promise as a novel therapeutic strategy for breast cancer, particularly for TNBC subtypes. Biomarkers in breast cancer with the potential to serve as targets for the design of spiegelmers are presented in Table 3 .

Unlike antibodies, aptamers are smaller in size, which enables them to penetrate solid tumors more effectively, exhibit longer retention, achieve uniform distribution, and avoid batch-to-batch variability. 92 , 128 , 129 Likewise the heterochiral creation of D-DNA with a cap from the L-DNA domain results in its stability of more than 24 hours in biological systems. 130 So spiegelmer does not require chemical modification for their stability, which makes them have the same physical and chemical properties as form D -nucleic acids. 131 In addition, the spiegelmers in in vivo have minimal immunogenicity. 36 An intriguing aspect of spiegelmers is their nuclear localization of L-DNA, in contrast to D-DNA, which is primarily localized in the cytosol and only transiently enters the nucleus before returning to the cytosol. 131 Danyang Ji and colleagues demonstrated that creating a circular L-RNA aptamer is an effective strategy to enhance both affinity and selectivity towards the target. 132

To solve the phenomenon of antigen escape that also occurs in breast cancer, studies have been directed towards the development of dual antibodies, but they have the challenge of being immunogenic. 65 Dual aptamer combines have the ability to bind to two targets in cells. 28 Therefore, evidence suggests that dual spiegelmers could be a excellent option for targeted delivery of chemotherapy agents.In fact, they can serve as an alternative to antibodies in the targeted delivery of chemotherapy agents, thereby reducing the side effects of these agents.

Moreover, designing spiegelmers to specifically target breast cancer biomarkers could enable personalized medicine strategies tailored to highly expressed genes and advancing the field of theranostics. Spiegelmers have advantages for targeting nuclear biomarkers through precise subcellular localization.Additionally, spiegelmers could enhance emerging treatments like PDT and PTT by improving the targeted delivery and tumor penetration of photosensitizers, and nanoparticles.Consequently, spiegelmers possess outstanding properties that make them highly promising candidates for targeted breast cancer therapy and theranostic in the future ( Figure 4 ).

10. Spiegelmer Aptamers and Breast Cancer Treatment

The CXCR4 receptor of cancer cells binds to the stromal-cell derived factor-1 (SDF-1) molecule secreted by cancer-associated fibroblasts, which activates signaling pathways associated with metastasis, drug resistance, and angiogenesis. Elevated CXCR4 expression in metastatic cell lines confirms its involvement in epithelial-to-mesenchymal transition (EMT) and breast cancer metastasis. in addition increased expression of SDF-1 in non-metastatic breast cancer cells was associated with progression to a cancer stem cells (CSC)-like phenotype and activation of the NF-κB pathway, followed by progression of EMT. Inhibition of CXCR4 is also associated with the induction of CDH1 gene expression. 114 , 115 Likewise, SDF-1/CXCR4 signaling is associated with lung metastasis and reduced survival in breast cancer. 117 Studies on CSC breast cancer have demonstrated that the SDF-1/CXCR4 pathway plays a crucial role in various biological processes, including the enhanced phosphorylation of proteins that regulate cell migration and invasion. 116 Therefore, a promising approach in breast cancer is to use a blocker of the SDF-1/CXCR4 signaling axis. Olaptesed Pegol or spiegelmer NOX-A12, an L-RNA conjugate of SDF-1, exhibits an extended half-life of 38 hours. Spiegelmer NOX-A12 has shown that it is effective in treating and sensitizing to chemotherapy and drugs in chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), multiple myeloma, colorectal cancer, pancreatic cancer, and glioblastoma, remarkably some treatments are in clinical trials. 44 - 49 Spiegelmer NOX-A12 has synergistic effects with anti PD1 antibodies by increasing T-cell migration to the tumor area, which could be an option for cancers that are resistant to ICI treatment. 50 PD-1 antibodies is already approved from TNBC subtype but patient acquired resistance to treatment. 133 Although spiegelmer NOX-A12 has not yet been established as a standard treatment for breast cancer, theoretical evidence suggests it holds promise as a therapeutic candidate. This is particularly relevant for TNBC, which NOX-A12 could be considered as part of combination therapy to enhance treatment efficacy and prevent to ICI treatment.

Sphingosine 1-phosphate (S1P) is a lipid mediator that is expressed at higher levels in breast cancer tissue compared to normal tissue, particularly in cases where the cancer has metastasized to lymph nodes. This elevated expression of S1P contributes to the proliferation, metastasis, lymphangiogenesis, and angiogenesis associated with breast cancer. 118 The use of anti-S1P antibody in a mouse model significantly reduced tumor volume, decreased VEGF expression, and inhibited angiogenesis, suggesting that targeting S1P is a promising strategy for anticancer therapy. 119 PEGylation NOX-S93 spiegelmer binds to S1P and effectively inhibits S1P mediated angiogenesis. 51 Therefore, this spiegelmer could be considered as a new treatment for breast cancer.

High-mobility group AT-hook1 (HMGA1) protein regulates gene expression by binding to DNA and assembling multi-protein complexes known as “enhanceosomes.”(123). HMGA1 expression is elevated in breast cancers negative for ER and PR and is correlated with poor prognosis and lymph node metastasis. 123 Silencing HMGA1 in TNBC cells inhibits migration and proliferation while promoting epithelial differentiation. 122 Additionally, treatment with an HMGA1-targeting antibody suppresses tumor cell migration and invasion. 120 Christian Maasch and colleagues developed a PEGylated NOX-A50 spiegelmer with an L-RNA structure. This spiegelmer binds to HMGA1 with high affinity and effectively reduces tumor size in a mouse model. Additionally, they demonstrated that using polyethyleneamine polyplexes enhances the retention time of the spiegelmer in the tumor region. 52 These findings suggest that HMGA1 may serve as a promising therapeutic target and potential adjuvant treatment in breast cancer.consideration PEGylated NOX-A50 spiegelmer as treatment option from breast cancer need to future studies.

The CC chemokine ligand 2 (CCL2) recruits monocytes to the tumor microenvironment, where tumor-associated macrophages (TAMs) subsequently release growth factors that promote tumor progression. Elevated levels of CCL2 are commonly observed in breast cancer, with high expression detected across cell lines representing four distinct breast cancer subtypes. Furthermore, CCL2 levels increase as the tumor advances. Inhibiting CCL2 with a specific antibody has been shown to effectively halt tumor growth and angiogenesis in mouse models. 124 - 127 PEGylated spiegelmer NOX-E36 (emapticap pegol) is a L-RNA aptamer that specifically binds to and inhibits the chemokine CCL2. It is currently being evaluated in a Phase IIa clinical trial for diabetic patients with albuminuria. 53 , 54 During TNBC, the infiltration of tumor-infiltrating lymphocytes (TILs) decreases while the presence of TAMs increases. A study in cancer-bearing mice demonstrated that pretreatment with the spiegelmer NOX-E36 prior to immune checkpoint inhibitor (ICI) therapy resulted in a significant reduction in tumor volume. 55

Evidence indicates that the development of spiegelmers, with their unique properties, has the potential to revolutionize breast cancer treatment ( Figure 5 ). Integrating spiegelmer-based therapies with current treatments and immunotherapies may enhance overall patient outcomes.

11. Challenges and Future Direction

Despite their superior biostability and low immunogenicity, Spiegelmers face several significant limitations that hinder their broader clinical adoption. Their inherent bioorthogonality, while conferring nuclease resistance, precludes enzymatic amplification, sequencing, and manipulation using standard molecular biology tools, necessitating labor-intensive solid-phase synthesis and indirect selection-reflection SELEX approaches that require inaccessible enantiomeric targets. Furthermore, the stereospecificity of Watson–Crick base pairing prevents direct hybridization with endogenous D-nucleic acids, limiting applications in antisense technologies without complex chimeric or intermediary designs. 134 Although extracellular Spiegelmers exhibit favorable pharmacological profiles, intracellular delivery remains challenging, and emerging evidence indicates that certain L-RNA sequences particularly G-rich motifs can induce cytotoxicity and innate immune activation via toll-like receptors, underscoring the need for systematic characterization of sequence-dependent intracellular behaviors. Addressing these challenges through the development of cross-chiral enzymatic tools, rational design principles for heterochiral interactions, and comprehensive interactome mapping will be critical to unlocking the full therapeutic potential of L-oligonucleotides. 135

Among other challenges, one can cite the inherent synthetic constraints of Spiegelmer production: their SELEX-based selection necessitates polymerases capable of incorporating L-nucleotides enzymes absent in nature thereby imposing reliance on solid-phase phosphoramidite chemistry and the laborious preparation of enantiomeric targets, which collectively limit oligonucleotide length, synthetic fidelity, and target diversity. 33 Although engineered mirror-image polymerases (e.g., Dpo4, Pfu, T7) and cross-chiral variants represent promising enzymatic alternatives, their scalable production and practical implementation remain active areas of investigation. 136

Additionally, the lack of WC base pairing between nucleic acids and spiegelmers limits their nucleic acid targets in diagnostic and therapeutic applications, while they remain targets for oligonucleotide antisense therapies, although this characteristic may offer some advantages. Current approaches to overcome this include the use of peptide nucleic acid intermediates and chimeric D/L nucleotides to facilitate spiegelmer binding. Finally, there is a scarcity of studies on spiegelmer behavior within biological environments, representing another area in need of further investigation. 131 A recent study demonstrated that single-stranded G-rich L-RNA sequences interact with numerous proteins containing RNA recognition motifs (RRMs) and participate in mRNA processing and splicing; however, 2′-O-methyl modifications can reduce these interactions and prevent the toxicity of spiegelmers in biological systems. 137

The use of spiegelmers in therapy can be associated with side effects; for instance, the NOX-A12 spiegelmer has been linked to inhibition of hematopoiesis. 138 Nevertheless, the stability of spiegelmers, combined with recent advances in targeted breast cancer therapy using scaffold-based delivery systems, offers solutions to many of these challenges. This targeted drug delivery method enables controlled release of therapeutics directly at the tumor site, thereby minimizing systemic side effects. Moreover, these scaffolds can be adapted in various forms and combined with different drugs for post-surgical treatment, enhancing targeted therapy effectiveness and reducing the risks of metastasis and recurrence. 139 , 140

12. Conclusion

The heterogeneous nature of breast cancer has driven the development of diverse biomarkers for both diagnosis and targeted therapy. Spiegelmers, a class of XNA aptamers, differ from conventional aptamers by possessing an L-nucleotide backbone rather than relying on chemical nucleotide modifications for stability. Although their synthesis is challenging and their range of targets limited, spiegelmers bind with high affinity to key biomarkers such as proteins and RNA. Their small size confers excellent tumor tissue penetration and low immunogenicity. Conjugation of spiegelmers with chemotherapeutic drugs, photosensitizers, and nanoparticles underpins novel breast cancer treatments involving targeted drug delivery, PDT, and PTT. Post-surgically, spiegelmers can be co-delivered with drugs via scaffolds to prevent recurrence and metastasis while minimizing side effects. Additionally, their ability to bind fluorescent dyes and radioisotopes makes them well-suited for real-time intraoperative imaging to delineate tumor margins. Overall, spiegelmers represent promising candidates for the development of breast cancer theranostics.

Abbreviations

Estrogen receptors

Progesterone receptors

Human epidermal growth factor receptor 2

Triple-negative breast cancer

Immunohistochemistry

Photodynamic therapy

Food and drug administration

Positron emission tomography

Magnetic resonance imaging

Xeno nucleic acids

2′-hydroxyl of ribose

Systematic Evolution of Ligands by Exponential Enrichment

Restricted Boltzmann Machines

B cell acute lymphoblastic leukemia

Chimeric antigen receptor T

gonadotropin-releasing hormone

Circulating tumor DNA

Circular RNAs

Bisphenol A

Insulin-like growth factor-1

Sex hormone-binding globulin

CC chemokine ligand 2

Tumor-associated macrophages

Tumor-infiltrating lymphocytes.

Author Contributions: Y.A.H., A.S., and R.A., and R.N., wrote the main manuscript text. A.S. edited the manuscript. Finally, all authors reviewed the manuscript. All authors read and approved the final manuscript.

Funding: The authors received no financial support for the research, authorship, and/or publication of this article.

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

AI-Assisted Text Generation: During the manuscript preparation, the authors utilized [ChatGPT/GPT] solely for linguistic enhancement and initial structuring of non-technical sections. All AI-processed content was critically evaluated, rewritten, and scientifically validated by the authors to ensure compliance with academic standards and originality.

ORCID iD

Ali Shojaeian https://orcid.org/0000-0002-1166-385X

Ethical Considerations

This study was approved by the ethics committee of Hamadan University of Medical Sciences (IR.UMSHA.REC.1404.718). It is worth noting that the scientific code of this project is 140408277925.

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