Unlocking Cancer's Code: The Future of DNA Nanotechnology

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USE OF DNA NANOTECHNOLOGY IN CANCER DIAGNOSIS AND THERAPY Abstract: DNA nanotechnology has emerged as a promising tool in cancer management, offering innovative solutions for diagnosis, therapy and monitoring. This review paper provides a comprehensive overview of the current state of DNA nanotechnology in cancer research, highlighting its potential applications, challenges, and future directions. We discuss the use of DNA nanoparticles as contrast agents for imaging techniques, DNA-based biosensors for detecting cancer biomarkers and nanoparticle-mediated delivery of genetic material for cancer diagnosis. Additionally, we explore the role of DNA nanotechnology in cancer therapy which includes targeted gene therapy, DNA-based immunotherapy and combination therapies. The integration of DNA nanotechnology with biomedical instruments such as microfluidic devices and biosensors has also been discussed. Finally, we address the challenges and future directions in this field, including toxicity and biocompatibility concerns, scalability and manufacturing challenges and emerging trends and future perspectives. The potential of DNA nanotechnology to revolutionize cancer management is vast with possibilities for early detection, personalized treatment, and improved patient outcomes. However, further more research is needed to overcome the existing challenges and to fully realize the benefits of DNA nanotechnology in cancer research and clinical applications. This review aims to provide a comprehensive resource for researchers and clinicians, inspiring future investigations and collaborations that harness the power of DNA nanotechnology to transform cancer treatment. By exploring the cutting-edge advancements in this field, we can unlock new possibilities for cancer diagnosis, therapy and management, ultimately improving the lives of cancer patients worldwide. I.IntroductionDNA nanotechnology involves designing and creating artificial nucleic acid structures for various applications. In this field, nucleic acids are being used as non-biological engineering materials for nanotechnology, rather than their traditional role as carriers of genetic information in living organisms. This area leverages the predictable self-assembly of DNA oligonucleotides to design and construct novel and highly precise nanostructures1 . These meticulously organized DNA motifs provide an ultra-fine framework for the next generation of nanofabrication, with potential applications in biotechnology, biomedicine, and beyond2 . Manipulating DNA at the nanoscale exploits its unique properties to produce individual molecules including DNA, RNA or proteins3 . Cancer diagnosis and therapy have numerous challenges that impact treatment effectiveness and patient outcomes. Disparities in access to oncologic imaging, pathology expertise, and technologies can affect the accuracy and timeliness of diagnoses, while inadequate communication and collaboration among oncologists, pathologists, and radiologists can lead to inefficiencies in care. Traditional cancer treatments such as chemotherapy, radiation therapy, targeted therapy, and immunotherapy have limitations, including lack of specificity, cytotoxicity, and multi-drug resistance, which pose significant challenges for favorable outcomes4 . Ethical dilemmas arise in clinical trials, quality assurance for surgery, radiotherapy, and medication, along with psychological challenges for patients, physicians, and caregivers. Despite rapid advances in cancer research and new diagnostic testing approaches transforming the landscape of diagnosis and care, integrating these innovations into clinical practice remains challenging. DNA nanotechnology offers several advantages in cancer therapy and diagnosis, making it a promising approach. It enhances early diagnosis through in vitro assays that detect cancer earlier than traditional methods and improves imaging capabilities for more precise detection and monitoring of cancer progression. This technology also refines targeted therapy by enabling more accurate drug delivery to cancer cells which minimizes the impact on healthy cells and also reduces systemic toxicity which leads to better patient outcomes with fewer side effects5 . Nano-theragnostic particles facilitate the detection and selective destruction of cancer cells simultaneously, combining diagnosis and treatment. Additionally, the development of advanced nanomaterials is a promising method for early cancerdetection, diagnosis and imaging6 . The history and evolution of DNA nanotechnology can be traced back to the early 1980s when Nadrian Seeman laid its conceptual foundation. Initially, DNA nanotechnology garnered widespread interest in the mid-2000s when DNA started being utilized as non- biological engineering material for nanotechnology rather than solely as genetic information carriers in living cells. The field experienced rapid growth in the 1990s, partly due to the industrial availability of chemically synthesized DNA molecules with arbitrary sequences. This era introduced functional nucleic acids (FNA) into DNA nanostructures enabling versatile applications such as biosensing, nanoplasmonics, and nanorobotics7 . In the past decade, structural DNA nanotechnology has made significant advancements which allowed researchers to order almost all necessary components, including modified DNA bases like biotinylated ones8 . II.DNA Nanoparticles for Cancer DiagnosisDNA nanoparticles are significantly advancing in cancer diagnosis by overcoming the limitations of old conventional diagnostic methods. Early detection of cancer is crucial for successful treatment, but existing methods often lack the necessary sensitivity and specificity. Nanotechnology with its high sensitivity, specificity and capacity for multiplexed measurements offers promising solutions. DNA nanoparticles when combined with other nanoscale materials like nanowires, gold nanoparticles, and quantum dots show great potential for early cancer diagnosis and timely therapy. They capture extracellular cancer biomarkers such as cancer-associated proteins, circulating tumor DNA and exosomes and enhance in vivo imaging for non-invasive diagnostics9,10. Despite these advancements' challenges remain in executing these technologies into clinical practice. Nonetheless the high surface area-tovolume ratio of nanoparticles and their ability to be specifically coat with antibodies and ligands significantly improve biomarker detection making nanotechnology-based diagnostic technologies real-time, convenient, and costeffective6 . 1. DNA nanoparticles as contrast agents for imaging techniquesDNA nanoparticles are being used as contrast agents in various biomedical imaging techniques due to their unique properties, these nanoparticles can be designed with specific features suitable for imaging modalities such as fluorescence imaging MRI, CT, ultrasound, PET and SPECT11. They offer advantages over conventional contrast agents including enhanced chemical and photostability of nanoparticle fluorophores, which result in better imaging quality and more accurate diagnoses12. DNA nanoparticles also improve detection limits, allowing for the visualization of biological processes at a much finer scale12. The development and application of these nanoparticles are advancing both current and emerging clinical bioimaging techniques, with the potential to significantly enhance diagnostic capabilities12. Additionally, they are used in photoacoustic imaging as exogenous contrast agents for cancer theranostics aiding in diagnosis, monitoring cancer progression, tracking metastasis, guiding tumor resection and facilitating drug delivery13 . DNA nanoparticles are being explored as MRI contrast agents due to their potential to enhance imaging quality, these nanoparticle-based contrast agents are characterized by their physio-chemical and magnetic properties as well as in vitro and animal studies, MRI studies and toxicity profiles14. Iron oxide nanoparticles (IONPs) have been extensively studied as MRI contrast agents due to their high biocompatibility and excellent magnetic properties providing an alternative to conventional agents15. Advances in magnetic nanoparticlebased MRI contrast agents include blood pool contrast agents biochemically targeted agents, stimulus-responsive agents, and ultra-high field MRI agents which are also used for cell labeling and tracking16. Additionally, magnetic nanoparticles (MNPs) have been investigated as T1-T2 dual-mode MRI contrast agents in both in vitro and in vivo biomedical preclinical studies with some already in clinical use17 . Nanoparticles are being utilized as contrast agents in Computed Tomography (CT) to enhance imaging quality. Metallic nanoparticles have been evaluated for their potential as X-ray CT contrast agents demonstrating in vivo contrast enhancement, targeted drug delivery, and dual/multipurpose imaging capabilities18. Researchers are increasingly focused on developing nanoparticulate CT contrast agents to overcome the limitations of current agents aiming to improve contrast enhancement and targeting capabilities19 . Cerium oxide nanoparticles (CeO2 NPs) have been proven effective as CT contrast agents due to their high X-ray attenuation, excellent biocompatibility, and radioprotective properties20. The development of nanoparticles as nextgeneration CT contrast agents is accelerating driven by the growing importance of CT in biomedical imaging21 . Positron Emission Tomography (PET) is a highly sensitive and non-invasive imaging technique utilized in cancer diagnosis. It uses ionizing radiation for bioimaging through the spontaneous emission of positrons from radionuclides such as F-18 and carbon-1122. PET scans typically involve a radioactive form of sugar which is absorbed by body cells at different rates based on their growth activity. Since cancer cells grow rapidly, they tend to absorb larger quantities of this sugar compared to normal cells. In the terms of DNA nanoparticles, PET imaging can be enhanced by radiolabeled theranostic nanoplatforms enabling quantitative cancer diagnosis and personalized therapeutic treatments using the same platforms23. These nanoplatforms leverage nanoparticles and the "enhanced permeation and retention" (EPR) effect to create innovative systems for PET imaging9. Fluorescence microscopy is an optical imaging technique that uses fluorophores that absorb light at one wavelength and emit it at a longer wavelength, this method involves labeling a sample with fluorophores such as DNA nanoparticles and illuminating it with specific wavelengths of light. The fluorescent molecules then emit light, which is captured by the microscope. This emitted fluorescence provides detailed information about the sample's structure, localization and interactions, this technique is invaluable for visualizing cellular components like DNA, proteins, and organelles, studying dynamic processes within living cells, detecting specific molecules such as cancer biomarkers, and achieving super-resolution imaging. It offers several advantages like noninvasiveness, high sensitivity and compatibility with living cell imaging though it is limited by photobleaching, phototoxicity and shallow penetration depth. DNA nanoparticles enhance this technique by serving as contrast agents with high specificity, biocompatibility and multifunctionality. They can be designed for specific targeting and also these are well-tolerated in biological systems and can carry payloads like drugs or imaging agents. In fluorescence imaging DNA nanoparticles labeled with fluorophores enable visualization of cellular processes and they also enhance MRI contrast and enable photoacoustic imaging by absorbing light and emitting acoustic signals12 . 2. DNA-based biosensors for detecting cancer biomarkersDNA-based biosensors are leading the way in cancer diagnostics providing exceptional sensitivity and specificity in identifying cancer biomarkers, these biosensors use DNA aptamers, which are short single stranded DNA molecules that selectively bind to target proteins or molecules linked to cancer. DNA based electrochemical biosensors use these aptamers to generate an electrical signal upon binding to their target offering a quantitative measurement of biomarker concentration. Additionally, DNA based florescence biosensors employ fluorescent tags that illuminate when the aptamer binds to its target, enabling visual detection of cancer biomarkers. DNA aptamers are single-stranded DNA molecules that can bind to those goals with high affinity and specificity, making such molecules precious in utilizing them for the building of DNA nanotechnology. Aptamer-functionalized DNA nanostructures are used in biosensing, bioimaging, cancer therapy and biophysics by combining the aptamers specificity with structural versatility of DNA nanostructure for different biological application24. Detailed methodologies exist for synthesizing and characterizing these aptamer-enabled DNA nanostructures, which are applicable to various designs in biomedical nanotechnology25. Often referred to as chemical antibodies due to their binding capabilities, DNA aptamers enable DNA nanotechnology for bioanalysis and cancer therapeutics by targeting specific molecules26. Additionally, aptamer-integrated DNA nanostructures are advancing in bio-sensing, bioimaging, targeted drug delivery, bioregulation, and biomimicry applications 27 . DNA-based electrochemical biosensors represent a significant research area in DNA nanotechnology offering sensitive and specific detection of biological targets. These biosensors leverage DNA’s properties such as self-assembly, programmability and precise molecular recognition, leading to innovative designs with enhanced sensitivity and specificity28. Research has focused on various aspects, including the construction and function of these biosensors, incorporating sensing materials, immobilization probe chemistries, hybridization conditions, and transducing principles29. Advances in DNA framework-engineered interfaces and multifunctional DNA nanostructures have been pivotal in creating stable and reproducible biosensors30. Notably, the development of electrochemical DNA biosensors for microRNA detection is progressing rapidly, with expectations of commercial availability soon driven by their high sensitivity and specificity31 . DNA-based optical biosensors are very important for early cancer detection these leveraging light to identify biological molecules with high sensitivity and without the need for labels. Key techniques include plasmonic waveguides which utilize surface plasmon resonance (SPR) to detect cancer biomarkers, photonic crystal fibers which enhance sensitivity by guiding light through periodic structures, slot waveguides, which confine light in tiny channels for precise detection, and metamaterials, engineered with unique optical properties. Traditional optical methods such as colorimetric techniques, optical coherence tomography, surface-enhanced Raman spectroscopy, and reflectometric interference spectroscopy are employed alongside advanced optical techniques like colorimetry for exosomal biomarkers, SPR for binding interactions, fluorescence using fluorescent labels and Raman scattering for analyzing scattered light32. A notable advancement in this field is the development of a three-dimensional electrochemical DNA biosensor utilizing 3D graphene-Ag nanoparticles to detect CYFRA21-1 in non-small cell lung cancer, exemplifying how biosensors are revolutionizing early cancer diagnosis 33 . 3. Nanoparticle-mediated delivery of genetic material for cancer diagnosisNanoparticle-mediated delivery of genetic material represents a cutting-edge approach in cancer diagnosis and therapy. Nanodiamonds are also being explored for their potential in cancer treatment serving as a multifunctional platform for delivering genetic factors and protein medication due to their specific physical and chemical properties34. Additionally, various cell-based delivery systems utilize cells like blood, immune, cancer, stem cells, sperm, and bacteria combined with nanoparticles have been developed to enhance targeted delivery for cancer therapy and diagnosis aiming to increase precision and minimize off-target effects35. Engineered nanoparticles play a crucial role in targeted drug delivery, directly transporting therapeutic agents to cancer cells, thereby improving drug efficiency and reducing side effects which is central to precision cancer medicine. Moreover, advancements in nanotechnologymediated cancer diagnosis are rapidly progressing, promising early detection, prediction, and prevention of cancer during its premalignant stages36 . 4. DNA nanoparticle-based point-of-care diagnosticsDNA nanoparticle-based point-of-care diagnostics have emerged as a developing solution for early cancer detection, harnessing advancements in various technologies to enhance clinical results. Paper-based point-of-care diagnostics offer an economical and user- friendly approach addressing challenges of delayed symptomatic detection and non- compliant sample collection inherent in traditional proteomics and genomic techniques. Innovations in paper chemistry, diverse assay types and enhanced detection techniques have significantly improved sensitivity and reliability often uses advanced nanomaterials for signal enhancement37. Nanozymes play a crucial role in reinforcing, miniaturizing, and enhancing the performance of these diagnostics meeting the ASSURED criteria (affordable, sensitive, specific, userfriendly, rapid, robust, equipment-free, and deliverable)38. Furthermore, biosensing chips, bolstered by nanoscience and material design, facilitate in situ and portable sensing ultimately improving clinical outcomes and reducing mortality rates39 . 5. Integration of DNA nanotechnology with biomedical instrumentsDNA nanoparticle-based point-of-care diagnostics represent a promising frontier for early cancer detection and also harnessing the unique properties of programmable and multifunctional DNA nanomaterials. These nanoparticles offer sub nanometer-level precision allowing for specific modifications with various chemical and biological entities, making them ideal for biosensing, bioimaging, and targeted delivery. Engineered DNA nanomaterials demonstrate highly specific capabilities for disease diagnosis and treatment with successful applications both in vitro and in vivo40. Despite of significant progress challenges like scalability, reliability, and safety in clinical applications remain. DNA nanotechnology combined with other nanoscale materials like nanowires, nanotubes, nanosheets, polymers, gold nanoparticles, quantum dots and iron oxides show great potential for early cancer diagnosis and timely therapy10 . Also, integrating DNA aptamers with DNA nanostructures enables the creation of functional nanomaterials which facilitate intelligent bioanalysis and clinical diagnosis through molecular computation. An example of this is the aptamer LXD-11b, which can recognize nucleated red blood cells in maternal peripheral blood26 . Microfluidic devices, lab-on-a-chip (LOC) systems, and biosensors/biochips are revolutionizing research which enables precise and efficient studies of cancer cell behavior, drug responses and metastasis. Microfluidic devices manipulate small fluid volumes within miniaturized channels which creates tumor-on-achip models that replicate the tumor microenvironment which facilitate highthroughput drug screening, and enabling liquid biopsies to detect circulating tumor cells or cancer-related biomolecules in bodily fluids41. LOC systems integrate multiple laboratory functions onto a single chip which supports pointof-care diagnostics, biomarker detection, and targeted drug delivery. Biosensors including electrochemical biosensors and immuno-biochips detects specific cancer biomarkers with high sensitivity and specificity which aids in early cancer diagnosis, personalized treatments and nanomedicine evaluation39 . III. DNA Nanoparticles for Cancer TherapyDNA nanoparticles have been recognized as a potential platform for cancer therapy and diagnosis-assisted treatment. The DNA-mediated nanostructures above have shown great impacts in cancer biology and they can bind to target proteins specifically expressed on the surface of cancer cells for early diagnosis and be used as a drug delivery carrier. These bio-templates may allow them to arrange organic, inorganic and biomolecules into different morphology with improved therapeutic applications42. Nanoparticle-based therapeutics stimulate tumor cell death, neo-antigen release and up-regulate antigen presentation and T cells activation in tumors which leads to improvement in cancer immunotherapy outcome by delivering pro-immune agents along with pro-inflammatory pathways into the tumor microenvironment. In a novel method, high-molecular-weight DNA has been employed as the carrier for biocompatible Mn^2+ loading and found to be very efficient chemo dynamic therapy by relying solely on endogenous H2O2 from cancer cells42 . 1. DNA nanoparticles as drug delivery systemsDNA nanoparticles are emerging as a promising tool in targeted drug delivery systems, complementing various organic and inorganic nanoparticle-based methods, such as magnetic nanoparticles and stimuli-responsive and biodegradable polymeric nanoparticles43. The field of nanomedicines and nano-based drug delivery systems is advancing rapidly, with nanomaterials enhancing the efficacy of both novel and existing drugs and contributing to selective diagnosis through disease marker molecules44. Significant progress has been made in the preparation and application of drug and gene co-delivery systems, addressing the challenges and future perspectives in designing and fabricating advanced delivery mechanisms45. Nanoparticles, including DNA nanoparticles, are utilized in biomedicine for their beneficial biological effects, with some nanoparticle- based drugs already approved by the U.S. Food and Drug Administration (FDA) for medical use46. Engineered nanoparticles are crucial in drug delivery systems, offering various nanocarriers developed for efficient drug transport47 . DNA nanoparticles are utilized in chemotherapy also to enhance the precision and effectiveness of cancer treatment. They work by loading chemotherapeutic drugs into DNA based nanocarriers, such as DNA tetrahedrons which can penetrate the cell membranes with minimal cytotoxicity thus improving drug delivery48. These nanoparticles can be functionalized to specifically target cancer cells which ensures that the chemotherapy drugs are delivered directly to the tumor site which results in reducing side effects and increasing treatment efficacy49. Also, nanoparticles protect the drugs from premature elimination and enhance the solubility of insoluble drugs, improving their pharmacokinetics50. By accurately targeting tumor cells while sparing healthy cells, nanoparticle-based drug delivery systems can also reduce the likelihood of developing drug resistance51 . DNA nanoparticles are employed in gene therapy to enhance treatment precision and effectiveness. These nanoparticles can be functionalized to deliver drugs or genes selectively to specific tissues or cells, crucial for precise targeting in gene therapy52. They can simultaneously load multiple drugs and genes which improves the effectiveness of each component and achieving synergistic effects in cancer therapy and pest management46. With the advent of RNA interference (RNAi) technology, nucleic acids can be delivered to target sites to replace or correct defective genes or knock down specific genes for therapeutic purposes53. Also, the combined drug delivery approach using nanoparticles is more effective in overcoming multidrug resistance in cancer cells. Immunotherapy harnesses the body's immune system to identify and attack cancer cells which involves key processes like antigen presentation, T cell activation and tumor cell destruction. Various forms, including immunetargeted antibodies, tumor vaccines, cellular immunotherapy, oncolytic viruses and cytokine therapy form the crux of this approach54. Nanodrug Delivery Systems (NDDS) which encapsulates drugs within nanoparticles which offers significant advantages such as precise tumor targeting, enhanced stability and biocompatibility, prolonged drug action and reduced toxicity55. These systems enhance immunotherapy efficacy by improving antigen presentation, T cell activation, and tumor cell killing55. Recent advancements in NDDS-based strategies include improved delivery of tumor antigens via cancer vaccines, the use of immunostimulatory materials to boost immune responses and the enhancement of T cell therapies. Furthermore, NDDS can induce immunogenic cell death, attracting immune cells to the tumor microenvironment, thereby amplifying the anti-cancer immune response. 2. Targeted gene therapy using DNA nanoparticlesRNA interference (RNAi) is a biological process where small non-coding RNAs (ncRNAs) interfere with the translation of target mRNA transcripts which results in gene silencing. In targeted gene therapy using DNA nanoparticles, double-stranded small interfering RNA (siRNA) therapeutics which is composed of 21-22 nucleotides are delivered into cells using targeted nanoparticles. Once these enter inside the cell siRNA induces sequence-specific enzymolysis of the target mRNA through complementary base pairing, suppressing gene expression56. RNA and RNA/DNA-based nanoparticles are engineered to conditionally activate RNA interference in human cells which allows for precise control over their formulation and function57. This process exploits doublestranded RNA (dsRNA) or small RNAs (sRNAs) to down-regulate target gene expression at the post-transcriptional level which leads to sequence-specific gene suppression. CRISPR/Cas9 gene editing, when combined with DNA nanoparticles enhances the precision and control in targeted gene therapy. This advanced delivery method enables gene editing on specific cell subsets within the body which potentially eliminates the need for invasive procedures. CRISPR-Cas9 facilitates targeted genomic excision of particular genes such as exon 80 in the COL7A1 gene through a dual-guide RNA sequence system delivered via DNA nanoparticles58. Nanomaterials enable controlled and targeted delivery of CRISPR/Cas9 with stimulus-sensitive techniques allowing for flexible release at the desired location for effective gene editing59. Researchers have also developed efficient delivery systems, such as lipid-encapsulated nanoparticles which guide CRISPR/Cas9 into the nucleus for precise gene editing60 . 3. DNA-based immunotherapy for cancer treatmentDNA-based immunotherapy is a cutting-edge cancer treatment that harnesses the body's immune system to combat the disease. DNA vaccines, using plasmid DNA are designed to activate the immune system against cancer which shows promising safety in early studies 61. This approach also includes targeted therapy which focuses mainly on proteins that influence cancer cell growth and spread, aligning with precision medicine principles by understanding genetic changes driving cancer. Additionally, CRISPR-Cas9 technology is employed to genetically modify patients' immune cells, such as T cells in order to enhance their cancer-fighting capabilities through precise genetic alterations62. As of July 31, 2023, the FDA has approved at least 23 different cancer Immunotherapeutics, by understanding the significant advancements and potential of immunotherapy in cancer treatment. IV. DNA Origami and Cancer Therapy1. DNA origami structures for drug delivery and imaging: DNA origami is an innovative technique for precisely folding long DNA chains into three dimensional nanomaterials which offers great potential in drug delivery and imaging due to their biocompatibility, ease of production and ability for site-specific chemical modifications. These structures can encapsulate drugs or genes for targeted delivery, releasing their payload in response to specific stimuli which thereby enhances treatment efficacy and reducing side effects63. Additionally, their precise design allows for the incorporation of imaging agents making them valuable for bioimaging applications and improving disease diagnosis63,64. In cancer therapy, peptoidcoated DNA origami can deliver anti-cancer drugs and target cell-surface receptors, while folate-functionalized DNA origami improves the targeting and efficacy of drug delivery to cancer cells65. The stability and precision of DNA origami enhance drug access to cancer cells which boosts treatment efficacy and reducing toxicity which makes them a focal point in medical research for overcoming drug delivery challenges66 . 2. DNA origami-based biosensors for cancer detectionDNA origami-based biosensors are cutting-edge tools for early cancer detection, offering high specificity and sensitivity in identifying cancer associated biomarkers. Researchers are developing DNA-origami-based nanorobots consisting of antibodies, aptamers and high brightness nanoparticles which are capable of carrying therapeutic and biochemical fingerprinting technologies for in cell theranostics which combines therapeutics and diagnostics. Additionally, a dynamic DNA origami book biosensor has been created to detect cancer- associated oligonucleotides and microRNAs (miRNAs). This biosensor features arrays of fluorophores as donors and acceptors along with fluorescence quenchers, producing a strong optical readout upon exposure to target molecules67 . 3. Potential applications of DNA origami in cancer researchDNA origami, a technique for folding DNA into nanoscale shapes and structures which has several promising applications in cancer research. DNA origami nanostructures (DONs) can serve as carriers for anti-cancer drugs and proteins which offers excellent editability, biocompatibility and in vivo stability which enhances drug delivery to cancer cells with increased precision and reduced toxicity66. Additionally, DNA origami can be used for imaging biological molecules and targeting cell-surface receptors implicated in cancer which aids in early detection and diagnosis. Its potential extends to tumor diagnosis where it can detect tumor markers and perform molecular imaging of tumors66 . Furthermore, researchers have demonstrated that octahedral DNA origami coated with specific peptides can prolong protein stability against proteolytic hydrolysis, benefiting drug delivery systems. 4. Integration of DNA origami with biomedical instruments for cancer therapyDNA origami is a compelling technique that facilitates the precise folding of long single-stranded DNA into various nanoscale structures, particularly suited for biomedical applications like cancer therapy. These structures known as DNA origami nanostructures (DONs) offers exceptional editability, biocompatibility and stability in vivo. DONs serve as effective nanocarriers for therapeutic delivery which enables the precise incorporation of anti-tumor medications. This capability enhances drug accessibility to cancer cells which leads to improvement in treatment efficacy and reduction in potential toxicity48 . Additionally, DNA origami allows for the controlled attachment of oligonucleotide molecular components with customizable sequences including imaging agents, drugs and therapeutic nucleic acids such as siRNA and mRNA. Such versatility in design provides multiple strategies for integrating these molecules into functional nanocarriers which broadens their potential applications in cancer treatment68 . 5.DNA origami-based nanoparticle-mediated drug deliveryDNA origami-based nanoparticle-mediated drug delivery represents a highly promising approach for advancing cancer therapy. This innovative method harnesses the precision of DNA origami which enables the construction of intricate 2D and 3D nanostructures through molecular self-assembly. These versatile nanoscale architectures provide exceptional control over geometry and can be meticulously functionalized with various molecules, including small molecule therapeutics, nucleic acids like antisense oligonucleotides or small interfering RNA, gene editing tools, and targeting ligands. By creating tailored drug delivery devices, DNA origami facilitates the precise delivery of therapeutic payloads to specific cells including cancer cells. Recent developments include enhancements in tunable drug release rates and strategies to bolster biological stability, underscoring the potential of DNA origami in advancing targeted drug delivery systems for cancer treatment68,69 . V. Challenges and Future Directions1.Toxicity and biocompatibility concernsToxicity and biocompatibility are crucial considerations in cancer therapy using DNA nanotechnology. DNA nanotechnology offers biocompatible solutions for cancer treatment, with nanoparticles often designed to reduce toxicity and enhance stability, aiding in precise targeting4. However, there are concerns about the potential toxicity of some nanomaterials, as they may exhibit low biocompatibility, weak targeting binding and difficulty in modification which can hinder their clinical application70. Nanocarriers can efficiently and selectively deliver nucleic acids to specific cells and tissues which improves the efficacy and reduces the toxicity of cancer therapeutics, especially in immunotherapy71 . Nanoparticle based drug delivery systems (DDSs) also enhance enhanced permeability and retention (EPR) effects which leads to passive tumor drug accumulation but the observed toxicity to normal tissues raises concerns about their real-world applications72. Cytotoxicity studies have shown that certain DNA nanoparticles possess significant anticancer effects with good biocompatibility, demonstrating a strong ability to kill cancer cells while causing minimal damage to non cancerous cells73 . 2. Scalability and manufacturing challengesScalability and manufacturing are significant challenges in applying DNA nanotechnology for cancer therapy. One major issue is the stability of DNA nanostructures within the body. Systematic studies are essential to understand their interactions with biological components to enhance in vivo stability. Additionally, the distribution and cellular uptake of DNA nanostructures are critical for effective treatment. The high cost of production of these nanostructures is another concern particularly for large-scale production and widespread clinical use74. In the realm of mRNA therapeutics advancements in biomaterials and delivery strategies face translational challenges that must be addressed to ensure clinical viability and the success of mRNA drugs in cancer treatment75. Improving drug delivery methods, especially those using polymeric nanocarriers is crucial for enhancing the efficacy of cancer therapy. Despite several nano-therapeutics being developed and some receiving FDA approval but further development is needed to tackle the remaining challenges in cancer nanomedicine76 . 3. Future directions: combining DNA nanotechnology with other cancer therapiesCombining DNA nanotechnology with other cancer therapies holds promise for more effective treatments. Future directions include the development of nanoparticle-based drug delivery systems for vaccinations, immunotherapy, biomarker detection, and imaging for various cancers with ongoing clinical trials indicating a bright future for these nanomedicines77. Advances in intelligent nanomaterials have significantly improved tumor- targeted drug delivery, immunotherapy, and temporospatial specific tumor imaging, enhancing our ability to address cancer's heterogeneous and complex nature78 . Nanomaterials are also being utilized in cancer immunotherapy which includes adoptive cell therapy (ACT), therapeutic vaccines and monoclonal antibodies with efforts focused on expanding their applications79. Progress in nanocarriers is being made for cancer diagnosis and treatment across various types, such as lung, breast, melanoma, colorectal, and bladder cancer50. Additionally, breakthroughs in nanoparticle-powered cancer vaccines are being explored for their potential and challenges which aims to inspire future immunotherapies that leverage nanotechnology for more effective and targeted treatments80 . 4. Addressing regulatory and ethical issuesAddressing regulatory and ethical concerns in DNA nanotechnology for cancer therapy and diagnosis is critical but the rapid advancements in this field present new challenges as public understanding of nanotechnology remains limited leading to misconceptions and unrealistic expectations. Despite their potential in disease diagnosis and drug delivery, nanoparticles possess unique properties that may pose risks to human health, such as generating reactive oxygen species and contributing to inflammatory diseases. Current exposure standards are inadequate, given the variability in nanoparticle manipulation and their potential long-term persistence in the environment. Issues extend beyond human health to include environmental impacts on agriculture and aquatic life as well as concerns about misuse in surveillance and security. Lessons from past technologies like genetically modified organisms highlight the importance of prioritizing safety and public trust through rigorous research and awareness campaigns to mitigate potential hazards effectively81 . 5. Emerging trends and future perspectives in DNA nanotechnology and biomedical instrumentsDNA nanotechnology has made significant strides in biomedical applications which offers new avenues for innovation. Researchers are developing scalable and programmable DNA nanoparticles that exhibit versatile functionalities. These nanomaterials are tailored for precise biosensing, bioimaging, and disease diagnosis, demonstrating efficacy in both laboratory settings and living organisms. They serve as sensitive biomarker detectors and enable targeted imaging of biological processes through precise modifications. Additionally, DNA nanomaterials show promise as carriers for therapeutic drugs, facilitating controlled release and enhancing applications in gene therapy and highcontrast imaging. Despite these advancements challenges like scalability, reliability and safety in clinical use persist, which prompt ongoing research efforts to fully harness the potential of DNA nanotechnology in biomedicine82 . 6. Overcoming biological barriers for DNA nanoparticle-mediated drug delivery- The field of DNA nanoparticle-mediated drug delivery for cancer therapy is advancing through research aimed at overcoming biological barriers. Reviews highlight the challenges of drug transport and underscore the necessity to address these barriers to effectively deliver nanotherapeutics to diseased sites. Innovative nanoparticle designs are crucial, as they play a pivotal role in enhancing drug delivery efficacy83. Insights from comprehensive reviews emphasize the significance of nanotechnology in precision cancer medicine, exploring various nanoparticle types and strategies for developing targeted nanomedicines across diverse cancer types. Additionally, recent advancements in cell-nanocarrier systems derived from stem cells, immune cells, and other sources are summarized, focusing on strategies to optimize drug delivery by overcoming biological obstacles in cancer treatment35 . Furthermore, discussions on targeted drug delivery in cancer using both organic and inorganic nanoparticle-based approaches highlight emerging technologies such as magnetic nanoparticles and stimuli-responsive polymeric nanoparticles, which are poised to improve therapeutic outcomes. VI. ConclusionIn this review, we have thoroughly examined the current state of DNA nanotechnology in cancer therapy and diagnosis, emphasizing its transformative potential in the field. The evidence gathered from various studies indicates that DNA nanoparticles, origami structures, and biosensors hold great promise for a range of applications, including imaging, drug delivery, gene editing, and immunotherapy. These technologies offer innovative solutions that could significantly enhance the precision and efficacy of cancer treatments. Despite the remarkable advancements, challenges and limitations persist, such as scalability, stability, and biocompatibility of DNA nanomaterials in clinical settings. Nonetheless, the collective findings from the reviewed studies underscore the substantial potential of DNA nanotechnology in revolutionizing cancer management. As research in this area continues to evolve, it is crucial to address these existing challenges, optimize current technologies, and explore new avenues for development. This review aims to serve as a comprehensive resource for researchers and clinicians, highlighting the exciting possibilities and inspiring future investigations and collaborations. By harnessing the power of DNA nanotechnology, there is a significant opportunity to transform cancer treatment, improve patient outcomes, and pave the way for novel therapeutic strategies in the fight against cancer.

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