Nucleic acid aptamers, often referred to as "chemical antibodies," are versatile, specific, and easily modifiable functional nucleic acids. There is a growing focus on new methods for the selection and target validation of aptamers, with the aim of expanding their biomedical applications in molecular diagnostics and therapeutics, which is currently a research hotspot. This review is composed of eight sections. In the first section, we briefly introduce aptamers and review their development in molecular diagnostics and therapeutics. The "Advantages of aptamers in molecular diagnosis and therapeutics" section summarizes and discusses the advantages of aptamers in these fields. The "New methods for screening aptamers" section presents and discusses nucleic acid aptamer screening methods, including both classical and novel approaches. In the "New methods for target validation" section, we explore new methods for target validation, covering aptamer structure validation, target recognition validation, and aptamer-target interaction validation. The "New methods for molecular diagnostics" section summarizes and discusses recent applications of aptamers in molecular diagnostics, particularly focusing on new mechanisms and detection strategies as well as their applications in various diseases. The "New methods for molecular therapeutics" section summarizes and discusses recent applications of aptamers in molecular therapeutics, emphasizing new mechanisms and aptamer-based therapy strategies, along with their therapeutic applications in different diseases. The "Challenges and future perspectives of nucleic acid aptamers" section addresses the challenges and future perspectives of aptamers in disease diagnosis and treatment. Finally, the "Conclusion" section shares our views on the future directions of aptamers in clinical disease molecular diagnostics and therapeutics.

Catalytic immunotherapy represents a promising approach to mitigate tumor metastasis and recurrence. The exploration of various advanced catalytic materials is opening new avenues for enhancing this therapy. However, metal-free carbon dot nanozymes with both NIR-II emission and NIR photoactivity are currently rare. Here, we report a metal-free multifunctional nanozyme, F-doped CDs (F-CDs) nanozyme, which exhibits a high NIR-II quantum yield and NIR-I photothermal effect for cancer catalytic immunotherapy. The F-CDs nanozyme demonstrated triplezyme-mimicking catalytic activity, including peroxidase (POD), glutathione peroxidase (GSH-px), and glucose oxidase (Gox). After modification with PEG, F-CDs@PEG exhibited excellent NIR-II bioimaging in vivo, comparable to FDA-approved dye indocyanine green (ICG). Under NIR laser irradiation, the remarkably enhanced catalytic activity of F-CDs@PEG disrupts the energy metabolism and redox balance of tumor cells, triggering intense tumor immunotherapy and reshaping the tumor immune microenvironment (TIME). Interestingly, we found that F-CDs@PEG can suppress immunosuppressive myeloid-derived suppressor cells (MDSCs) and alleviate T-cell exhaustion, leading to enhanced tumor growth inhibition and reduced splenomegaly. Importantly, F-CDs@PEG-mediated photothermal-enhanced catalytic immunotherapy can induce robust immune memory, offering long-term protection against tumor recurrence. Overall, the success of this study offers a feasible strategy for the future design and exploration of NIR-II CD-based nanozymes and metal-free nanocatalysts for catalytic immunotherapy.

We designed a Zn-doped NiB electrode on flexible filter paper via a mild electroless plating method (NiB–Zn@HP), the electrode has structural stability and high HER efficiency in a multi-pH system.

2D MoS2 holds significant promise for cancer therapy due to its unique physicochemical properties and biocompatibility. However, its precise effects in clinical colorectal cancer (CRC) remain poorly understood, as traditional cell-line evaluations often fail to reflect patient-specific tumor heterogeneity. To address this limitation, we developed a cancer patient-derived organoid platform for evaluating hydrophilic metallic MoS2 (M-MoS2) nanosheets in CRC. We established paired tumor and normal colorectal organoids from patient tissues and assessed them using 3D co-culture systems along with comprehensive analytical techniques, including electron microscopy and transcriptomics. Our findings demonstrated that M-MoS2 nanosheets selectively suppressed CRC organoid growth without affecting normal organoids, establishing a favorable therapeutic window. Mechanistically, M-MoS2 nanosheets were internalized by tumor organoids and localized primarily to mitochondria, inducing reactive oxygen species production and inhibiting the PI3K-AKT signaling pathway. This triggered G2-M cell cycle arrest and apoptosis specifically in tumor organoids. To our knowledge, this is the first report of 2D MoS2 in a cancer patient-derived organoid platform. Our cancer organoid-based platform provides compelling evidence of the efficacy and safety 2D M-MoS2 for CRC treatment, while offering a robust approach for the preclinical safety and functionality assessment of advanced biomaterials, thereby accelerating their practical applications and clinical translation.

Aptamers and aptamer-drug conjugates (ApDCs) have shown some success as targeted therapies in cancer theranostics. However, their stability in complex media and their capacity to evade lysosomal breakdown still need improvement. To address these challenges, we herein developed a one-step self-assembly strategy to improve the stability of aptamers or ApDCs, while simultaneously enhancing their delivery performance and therapeutic efficiency through a lysosome-independent pathway. This strategy involves the formation of stable complexes between disulfide monomer and aptamers (Sgc8) or ApDCs (Gem-Sgc8). Self-assembled Sgc8 NPs resisted nuclease degradation for up to 24 h, whereas the aptamer alone degraded within just 3 h. These self-assembled Sgc8 NPs, as well as Gem-Sgc8 NPs, demonstrated enhanced binding capabilities compared to Sgc8 aptamers or Gem-Sgc8 alone. Furthermore, lysosome-independent cellular uptake was significantly improved, which in turn increased the therapeutic efficacy of Gem-Sgc8 NPs by 2.5 times compared to Gem-Sgc8 alone. In vivo results demonstrated that Gem-Sgc8 NPs can effectively suppress the growth of tumors. The same self-assembly strategy was successfully applied to other aptamers, such as MJ5C and cMET, showing the generalizability of our method, Overall, this aptamer self-assembly strategy not only overcomes the limitations associated with instability and lysosomal degradation but also demonstrates its broad applicability, highlighting its potential as a promising avenue for advancing targeted cancer theranostics. STATEMENT OF SIGNIFICANCE: We developed a one-step self-assembly strategy to improve the stability of aptamers or ApDCs and enhance their drug therapeutic efficiency through a lysosome-independent pathway. The stability of self-assembled Sgc8 nanoparticles (NPs) was significantly improved. The resulting Sgc8 NPs or GEM-Sgc8 NPs exhibited enhanced binding ability compared to Sgc8 aptamers or GEM-Sgc8 alone, and they also facilitated lysosome-independent cellular uptake, resulting in a 2.5-fold increase in therapeutic efficacy of GEM-Sgc8-NPs. The same self-assembly strategy was successfully applied to other aptamers, such as MJ5C and cMET, showing the generalizability of our method.

Immunotherapy has revolutionized the treatment of lung cancer, but many patients still experience inadequate responses or develop resistance, emphasizing the urgent need for more effective therapeutic strategies. Here, we present the design of an inhalable flexible 2D molybdenum disulfide (MoS2) T cell hyperactivation platform (2D MoS2-THP) based on anchoring therapeutic proteins onto the surface of metallic molybdenum disulfide. By loading IL2 and anti-PD1 proteins onto a flexible metallic molybdenum disulfide crystalline form, we achieved effective T cell hyperactivation. Our platform leverages the synergistic effects of anti-PD1 and IL2 to remodel exhausted T cells while simultaneously overcoming regulatory T cell (Treg)-mediated immunosuppression, resulting in a superior T cell activation. Additionally, 2D metallic MoS2 not only serves as a carrier for delivering therapeutic proteins but also plays a pivotal functional role by inducing cuproptosis in Tregs through disruption of mitochondrial function and elevation of oxidative stress, thereby clearing a key barrier to effector T cell activation. The inhalation administration route further improves intratumoral accumulation, enhances therapeutic potency, and minimizes systemic immune-related side effects. Overall, this inhalable "three-in-one" immunotherapy platform achieves robust T cell hyperactivation with reduced systemic toxicity, efficiently removing immunosuppressive barriers, and remodeling the tumor immune microenvironment to overcome cancer immunotherapy resistance.

Cancer immunotherapy is often associated with immune-related adverse events (irAE), particularly in the lungs, which can lead to treatment discontinuation and negatively impact patient outcomes. In this study, we explore a spatiotemporally controlled cotreatment of lung cancer and pulmonary irAE using inhalable albumin-chaperoned ultrathin MnO2 nanosheets (MnO2@BSA). The MnO2@BSA exhibits efficient reactive oxygen species scavenging and NIR-II photothermal properties. The inhalation route enhances MnO2@BSA accumulation in the lungs and tumor tissue, offering localized treatment with reduced systemic toxicity. We demonstrate that ultrathin MnO2@BSA nanosheets can inhibit the formation of neutrophil extracellular traps (NETs) by reducing reactive oxygen species (ROS) in neutrophils, thereby alleviating inflammation associated with irAE. In animal models, inhaled MnO2@BSA reduced lung injury and inflammatory cell infiltration, while also decreasing pro-inflammatory cytokine levels, such as TNF-α, IL-1β, and IL-6. Simultaneously, MnO2@BSA displays strong photothermal properties under 1064 nm laser irradiation, effectively ablating tumors. This photothermal therapy also induces immunogenic cell death (ICD), promoting the reuse of activated CD8+ T cells to enhance antitumor immunity. These dual effects─tumor destruction and irAE mitigation─highlight MnO2@BSA's potential as a therapeutic platform for addressing the challenges of immunotherapy in lung cancer. We anticipate that this work could contribute to the development of inhalable metal-based nanomedicine for clinical transformation in tumor immunotherapy.

Immune checkpoint blockade therapy holds promise for inducing lung cancer regression, but its effectiveness is often limited due to the immunosuppressive tumor microenvironment (ITME). Ferroptosis can remodel the ITME and enhance immunotherapy, but traditional inducers exhibit weak immune modulation due to off-target toxicity and low drug concentrations at the tumor site, resulting in poor efficacy. To address these challenges, we explore an inhalable immune activation nanoplatform, named LMS, designed to enhance ferroptosis-mediated immunosensitization in lung cancer. LMS owns high ferroptosis inducer loading and multienzyme catalytic activity. By nebulization delivery, we demonstrate that LMS can fast and efficiently delivery Statins to lung cancer with minimal systemic toxicity. LMS effectively triggers an oxidative stress storm and lipid peroxidation burst to induce ferroptosis in tumor cells, while leveraging ferroptosis and dendritic cell maturation to synergistically enhance T cell immune responses, thereby reversing ITME. Our nanoplatform achieved efficient tumor suppression in an in-situ lung cancer model, demonstrating the great potential of the inhalable immune activation nanoplatform in ferroptosis-regulated cancer immunotherapy. This work paves a new path for safely and efficiently reversing the ITME in lung cancer, potentially accelerating the development of inhalable immune nanoagonists for clinical applications in cancer immunotherapy.

Regulating membrane protein abundance through Lysosome Targeting Chimera (LYTAC) holds significant promise in addressing various diseases. However, the precise structural control of LYTAC molecules and how to improve their treatment efficacy remain elusive. In this study, we develop a multifunctional phototriggered LYTAC platform, named PT-LYTAC, to enhance targeted protein degradation using a photoactive bispecific aptamer chimera (PBAC). PBAC is designed with a precise modular approach that integrates an NIR photosensitive molecule into a bispecific aptamer chimera. Taking advantage of the low molecular weight and easy synthesis of the DNA aptamers, PBAC can efficiently transport the therapeutically relevant membrane protein PTK7 to lysosomes for degradation through the lysosomal pathway. Moreover, our investigation reveals that the multifunctional PT-LYTAC platform, enabled by DNA aptamers, promotes protein degradation by modulating cellular autophagy. By the combination of targeted protein degradation and spatiotemporally controllable regulation of intracellular oxidative stress, the function of tumor cells can be significantly inhibited. Under NIR laser irradiation, PT-LYTAC completely suppresses colorectal cancer growth with just one dose and a single laser treatment, all without any apparent side effects. We anticipate that this novel PT-LYTAC will expand the use of DNA-based LYTAC drugs and provide a new dimension for targeted protein degradation.

The development of cost-effective and corrosion-resistant catalytic electrodes for chlorine/oxygen evolution reaction (CER/OER) in large-scale industrial applications is a significant challenge. Herein, the sol-gel method is employed to achieve a uniform coating of ruthenium (Ru) doping copper (Cu) on titanium sheet (Ru + 20 % Cu@Ti), and the highly efficient industrial grade stable Ti dimensional stable anode can be quickly constructed at 723.15 K for 2 h. Cu doping reduces the vacancy formation energy of surface oxygen, promotes additional lattice oxygen vacancy assisted hydrolysis dissociation pathway, improves the selectivity and specific activity of CER at high concentration doping, and reduces the binding energy of OER intermediates (e.g., *OH, *O, and *OOH) at adjacent Ru active sites. The overpotentials require to reach the current density of 10 mA cm- 2 for CER and OER were only 365 mV and 232 mV at the conditions of 5.0 M NaCl (pH = 7.0) and 1.0 M KOH + 0.5 M NaCl. More importantly, Ru + 20 %Cu@Ti demonstrates excellent stability, operates continuously for over 340h at industrial current density in neutral and alkaline electrolytes, and its strengthening life reaches 64 h, with ultra-low performance attenuation. Impressively, the designed applied electrode (8.0 cm x 15.0 cm) achieves long-term CER at 0.2-0.3 A cm- 2. Further industrial grade evaluation of CER shows that its chlorine extraction polarizability, enhances life and weight loss meet the requirements of industrial applications.

Developing promising biodegradable second near-infrared (NIR-II) immune agonists to overcome the insufficient immunogenicity of tumor cells and the poor immune response remains challenging. In this work, a biodegradable "2D genesheet" is explored for synergistic NIR-II photothermal immunotherapy. The 2D genesheet is large-scale prepared through flexible supramolecular self-assembly between metallic-MoS2 and siRNA. The 2D genesheet not only exhibits an excellent photothermal conversion efficiency up to 56% in the NIR-II window, but also features unique H2O2-responsive biodegradability and high biocompatibility. The 2D genesheet can efficiently deliver siRNA(siIDO) to tumor cells and downregulate indoleamine 2,3-dioxygenase 1 (IDO1) protein expression, exerting intrinsic immune activation and reversing IDO1-mediated immunosuppression. The spatiotemporal-controlled NIR-II phototherapy can elicit systemic immunity by evoking immunogenic cell death, promoting dendritic cell maturation, and increasing T cell infiltration. The bioactive material is able to efficiently inhibit primary and distant tumors, achieving a 100% tumor eradication rate. This study broadens the application prospects of biodegradable NIR-II immune agonists, overcoming the limitations of current immunotherapeutic modalities.

Targeting tumor metabolism reprogramming has demonstrated a synergistic antitumor effect in photodynamic therapy of triple-negative breast cancer (TNBC). However, such a combination therapeutic regimen has encountered challenges, such as limited photosensitizer bioavailability and severe drug toxicity. Herein, ultrasmall metal-organic frameworks (MOFs) nanodots (MSPC) that encapsulate metabolism inhibitors and mitochondria-targeted photosensitizers are designed and fabricated for synergistic photodynamic therapy (PDT) of TNBC. The MSPC exhibits an acidic-sensitive drug release, leading to glutathione depletion and mitochondrial respiration suppression. Significantly, MSPC substantially reduces intracellular adenosine triphosphate (ATP) levels by simultaneously disrupting oxidative phosphorylation and impeding aerobic glycolysis. Therefore, the glutathione depletion combined with metabolism inhibitor increases oxidative stress, which improves the efficacy of mitochondria-targeted PDT. Additionally, the increased retention of photosensitizers within tumors, facilitated by aggregation-enhanced retention (AER) effect, extends the time window for long-term fluorescence/photoacoustic imaging-guided PDT of TNBC. MSPC-sensitized PDT significantly suppresses tumor growth with a single-dose injection and repeatable PDT. In summary, these renal-clearable and aggregation-enhanced tumor-retained nanodots indicate the feasibility of overcoming resistance to reactive oxygen species induced by metabolic reprogramming, thus holding significant implications for boosting PDT of TNBC.

Triple-negative breast cancer (TNBC) is one of the most malignant cancer types, characterized by a lack of efficient diagnostic and treatment methods in clinical practice. The development of effective targeted diagnosis and treatment for TNBC has become an important research focus. Here, we report the first proof-of-concept evidence of a glycoaptamer dual-target radionuclide probe (GDRP) for positron emission tomography/computed tomography (PET/CT) imaging of TNBC. The GDRP was created through precise molecular engineering of the aptamer AS1411, combined with three mannose ligands. The GDRP exhibited significantly increased serum stability and a high affinity for TNBC cells through a dual targeting mode. The advantages of our developed GDRP were further demonstrated by its excellent in vivo PET/CT dynamic imaging performance, featuring a long imaging window and high spatiotemporal resolution. This flexible method allows for the preparation of glycosylated functional nucleic acid molecular probes with high serum stability and tumor specificity. We anticipate that this PET/CT molecular probe will expand the development of glycoaptamer-based radioactive drugs and provides molecular tools for the clinical diagnosis of TNBC.

Melanoma recurrence and full-thickness skin defects severely impair patient recovery and quality of life. There is an urgent need for therapeutic platforms that not only eliminate residual melanoma cells but also accelerate wound healing. Cuproptosis has emerged as a promising anticancer strategy. However, a significant challenge remains in overcoming the adaptive defenses of cancer cells and sensitizing them to cuproptosis. Herein, we explore a mitochondrial-targeted Ca/Cu dual-ion chaos inducer (Mito-chaos) by integrating a mitochondria-targeted curcumin derivative (MitoCur) into a calcium- and copper-co-doped Prussian blue nanoplatform. Mito-chaos exhibits excellent mitochondrial targeting, multienzyme-mimicking catalytic activity, and strong NIR-II photothermal properties. Mito-chaos can precisely deliver Cu2+ and Ca2+ ions into mitochondria, disrupting mitochondrial ion homeostasis, inducing calcium overload, and consequently amplifying cuproptosis. Combined with mild NIR-II photothermal treatment, Mito-chaos achieves effective melanoma suppression accompanied by robust antitumor immune activation. Beyond tumor eradication, Mito-chaos significantly enhances skin vascularization and collagen deposition, accelerating postoperative wound healing and reducing wound closure time. This mitochondria-targeted therapeutic platform not only effectively eliminates residual melanoma cells but also promotes tissue regeneration, providing an integrated and effective strategy for melanoma postoperative management. Our study presents a promising paradigm for precisely amplifying subcellular organelle dysfunction to boost cancer cuproptosis therapy and tissue repair.

Functional nucleic acids (FNAs), possessing specific biological functions beyond their informational roles, have gained widespread attention in disease therapeutics. However, their clinical application is severely limited by their low serum stability in complex physiological environments. In this work, a precise molecular programming strategy is explored to prepare glyconucleic acid aptamers (GNAAs) with high serum stability. Four glyconucleic acid modules compatible with commercial solid-phase synthesis are designed and synthesized. Through precise molecular design, the accurate modification of four different carbohydrate ligands at specific sites of DNA aptamers is achieved. It is demonstrated that glycosylation modification can significantly increase DNA aptamers' serum stability while maintaining their structures and high affinity. The stabilization effect is superior to that of currently commonly used commercial chemical modifications. Moreover, it is confirmed that this approach displays insignificant effects on the DNA aptamers' tumor-targeting ability and metabolism in vivo. This method offers a simple, economical, and efficient strategy for precise glycosylation modification of nucleic acids. This allows to prepare glycosyl functional nucleic acids with high serum stability, which can expand the application scope of functional nucleic acids and promote the practical transformation of functional nucleic acids.

Photodynamic therapy (PDT) is a clinically representative treatment strategy for cancer. However, conventional photosensitizers (PSs) are usually hampered by poor water solubility and low targeting capacity. Here, we report the precise molecular engineering of aptamer oligonucleotides to solubilize hydrophobic near-infrared (NIR) PSs for enhanced cancer PDT. Hydrophobic pyropheophorbide A (PA) is precisely conjugated to aptamer oligonucleotides by combing DNA solid-phase synthesis technology and Cu-free click chemistry. Precise coupling of insoluble PA to hydrophilic aptamer oligonucleotides vastly improves its solubility to as high as 750 mu M in water without any cosolvent, resulting in an enhanced NIR fluorescence property (quantum yield = 23%) and photoactivity. Moreover, the targeting ability of aptamer oligonucleotides is not affected by our molecular engineering strategy. Abundant reactive oxygen species (ROS) are produced intracellularly with 660 nm laser irradiation, eliciting mitochondria damage and cell death. Tumor growth is effectively inhibited with a single dose of aptamer-PA conjugates without in vivo toxicity. Their therapeutic effect is more than 20-fold higher than that of PA. Compared with traditional formulation, using aptamer oligonucleotides as functional carriers to solubilize hydrophobic NIR PSs is more precise and operable. Our DNA molecular engineering strategy paves a way for the rational design of molecularly targeted medicine for future clinical cancer therapy. [GRAPHICS] .

The combination of programmed cell death ligand-1 (PD-L1) immune checkpoint blockade (ICB) and immunogenic cell death (ICD)-inducing chemotherapy has shown promise in cancer immunotherapy. However, triple-negative breast cancer (TNBC) patients undergoing this treatment often face obstacles such as systemic toxicity and low response rates, primarily attributed to the immunosuppressive tumor microenvironment (TME). In this study, PD-L1-targeted theranostic systems were developed utilizing anti-PD-L1 peptide (APP) conjugated with a bio-orthogonal click chemistry group. Initially, TNBC was treated with azide-modified sugar to introduce azide groups onto tumor cell surfaces through metabolic glycoengineering. A PD-L1-targeted probe was developed to evaluate the PD-L1 status of TNBC using magnetic resonance/near-infrared fluorescence imaging. Subsequently, an acidic pH-responsive prodrug was employed to enhance tumor accumulation via bio-orthogonal click chemistry, which enhances PD-L1-targeted ICB, the pH-responsive DOX release and induction of pyroptosis-mediated ICD of TNBC. Combined PD-L1-targeted chemo-immunotherapy effectively reversed the immune-tolerant TME and elicited robust tumor-specific immune responses, resulting in significant inhibition of tumor progression. Our study has successfully engineered a bio-orthogonal multifunctional theranostic system, which employs bio-orthogonal click chemistry in conjunction with a PD-L1 targeting strategy. This innovative approach has been demonstrated to exhibit significant promise for both the targeted imaging and therapeutic intervention of TNBC.

Breast cancer (BC) poses a significant threat to women’s health, with triple-negative breast cancer (TNBC) representing one of the most challenging and aggressive subtypes due to the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. Traditional TNBC treatments often encounter issues such as low drug efficiency, limited tumor enrichment, and substantial side effects. Therefore, it is crucial to explore novel diagnostic and treatment systems for TNBC. Multifunctional molecular probes (MMPs), which integrate target recognition as well as diagnostic and therapeutic functions, introduce advanced molecular tools for TNBC theranostics. Using an MMP system, molecular drugs can be precisely delivered to the tumor site through a targeted ligand. Real-time dynamic monitoring of drug release achieved using imaging technology allows for the evaluation of drug enrichment at the tumor site. This approach enables accurate drug release, thereby improving the therapeutic effect. Therefore, this review summarizes the recent advancements in MMPs for TNBC theranostics, encompassing the design and synthesis of MMPs as well as their applications in the field of TNBC theranostics.

Nanomedicine holds promising potential for cancer therapy, yet challenges such as low active pharmaceutical ingredient (API), nanocarrier toxicity, complex preparation processes, and a lack of targeting ability hinder clinical applications. Here, we present a versatile method for preparing DNA aptamer-based full-API nanodrugs (ICS AFANDs). This approach involves the self-assembly of FDA-approved chemotherapeutics (irinotecan), photosensitive drugs (Chlorin e6, Ce6), and the DNA molecule of the Sgc8 aptamer. DNA aptamers are firstly confirmed to not only impart solubilization and targeting ability to ICS AFANDs but also play a pivotal role in engineering and stabilizing the formation of self-assembled nanodrugs. Our method not only significantly enhances the chemotherapy effectiveness of irinotecan by 102-fold, but also improves the near-infrared (NIR) fluorescence properties and photoactivity of Ce6. Combining photodynamic therapy (PDT) and chemotherapy (CT), our ICS AFANDs achieve complete suppression of colorectal cancer growth, with only one dose and one laser treatment, all without apparent side effects. Our strategy is a versatile method for preparing carrier-free and multifunctional DNA nanodrugs for synergistic tumor therapy. The smart and efficient full-API DNA nanodrug not only broadens the scope of synergistic tumor therapy but also facilitates the practical translation of DNA nanodrugs to clinical use.

Second near-infrared (NIR-II) carbon dots, with absorption or emission between 1000 and 1700 nm, are gaining increasing attention in the biomaterial field due to their distinctive properties, which include straightforward preparation processes, stable photophysical characteristics, excellent biocompatibility, and low cost. As a result, there is a growing focus on the controlled synthesis and modulation of the photochemical and photophysical properties of NIR-II carbon dots, with the aim to further expand their biomedical applications, a current research hotspot. This account aims to provide a comprehensive overview of the recent advancements in NIR-II carbon dots within the biomedical field. The review will cover the following topics: (i) the design, synthesis, and purification of NIR-II carbon dots, (ii) the surface modification strategies, and (iii) the biomedical applications, particularly in the domain of cancer theranostics. Additionally, this account addresses the challenges encountered by NIR-II carbon dots and will outline future directions in the realm of cancer theranostics. By exploring carbon-based NIR-II biomaterials, we can anticipate that this contribution will garner increased attention and contribute to the development of next-generation advanced functional carbon dots, thereby offering enhanced tools and strategies in the biomedical field.