transcription (IVT) using T7 RNA polymerase is a key step in mRNA synthesis for therapeutic applications. However, the generation of double-stranded RNA (dsRNA) byproducts during IVT─primarily due to RNA rebinding and self-priming─triggers innate immune responses and reduces translation efficiency. Here, we present a simple and effective strategy to minimize dsRNA formation during IVT by incorporating PEGylated graphene oxide (PEG-GO). Graphene oxide (GO) preferentially binds single-stranded nucleic acids, but its use is limited by protein adsorption and low solubility in Mg-containing buffers. PEG modification improves GO's dispersibility and reduces protein binding, allowing selective sequestration of nascent RNA without inhibiting T7 RNA polymerase activity. The addition of PEG-GO to the IVT reaction reduced the dsRNA content by over 75% while maintaining RNA yield and accelerating transcription kinetics. Moreover, mRNA synthesized in the presence of PEG-GO showed enhanced protein expression and reduced interferon-β secretion in transfected cells, comparable to post-IVT-purified mRNA. Our work demonstrates PEG-GO as a practical additive for improving the quality and scalability of IVT-based mRNA production.

The emerging field of glycoRNAs, RNA molecules covalently modified with glycans, challenges the long-held belief that glycosylation is exclusive to proteins and lipids. The discovery of 3-(3-amino-3-carboxypropyl) uridine (acp3U) as a specific N-glycan attachment site has been a major breakthrough, establishing glycoRNA as a structurally defined and functionally relevant biopolymer. This new function of acp3U suggests its crucial regulatory node that correlates translation with other cellular processes, such as immune modulation and cell signaling. The presence of glycoRNAs on the cell surface and their interaction with immune receptors imply their involvement in cell-to-cell communication. Furthermore, studies have begun to associate altered glycoRNA patterns with conditions like cancer and inflammation, opening up possibilities for diagnostic and therapeutic applications. Despite the rapid progress in this field, several key challenges remain, including the inherent bias of current detection methods, the difficulty of isolating pure glycoRNA samples from complex cellular mixtures, and the largely unknown mechanisms of specific glycan linkages. Future research must focus on developing unbiased and sensitive analytical technologies to accurately map these modification patterns at a single-nucleotide resolution. This review summarizes the chemical and enzymatic mechanisms of RNA glycosylation sites, highlights its potential functional roles in cells, and outlines future research aimed at uncovering its full biological and therapeutic potential.

CRISPR-Cas12a is a versatile biosensing platform that detects sequence-specific DNA or RNA targets via a CRISPR RNA (crRNA) guide. While Cas12a's specificity is dictated by its crRNA, chemical modifications within the crRNA can influence nuclease performance. Here, we examined the effects of two well-known RNA modifications, N-methyladenosine (mA) and 5-methylcytosine (mC), introduced into the different positions of the guide region of a crRNA. Melting temperature () analysis showed that mA had a minimal impact on RNA-DNA duplex stability. In contrast, the incorporation of mC residues stabilized the duplex. Using a fluorescence recovery assay, we found that both modifications preserved Cas12a's nuclease activity, indicating that small thermodynamic shifts in duplex formation are insufficient to disrupt its catalytic function. Despite the greater increase with mC, mA incorporation led to a faster fluorescence recovery rate than that with mC.

Chemical modifications in RNA therapeutics have addressed major challenges by enhancing metabolic stability, cellular uptake, and biological activity─regardless of their mechanism of action. Here, we report on the synthesis of 4'--ethynyl cytidine (4'--EthC) and its 2'--methylated derivative (4'--EthC-2'-OMe) as phosphoramidite building blocks and their subsequent incorporation into oligonucleotides. These ribose C4-terminal alkyne modifications provide a click handle directly within oligonucleotides. The novel modification is accessible via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and serves as a universal 4'--ribose modifier on the oligonucleotide level. We identified both aromatic and aliphatic triazole residues that increase the thermodynamic stability in A-form RNA duplexes. Furthermore, 4'--triazole-modified oligonucleotides exhibit high resistance to nuclease-mediated degradation in metabolic stability assays. Finally, we introduced the novel modification and its substituted triazoles into guide RNAs (gRNAs) for site-directed A-to-I editing in mammalian cells and compared their performance with phosphorothioate-modified gRNAs.

The heterogeneous and insufficient reactive oxygen species (ROS) level in the tumor microenvironment (TME) limits the effectiveness of conventional ROS-responsive drug delivery systems (DDSs). To overcome this, a pH/ROS dual-responsive amphiphilic copolyprodrug (P(CA-DOX-Fc)-PEG) was designed by integrating cinnamaldehyde (CA), doxorubicin (DOX), and ferrocene dicarbohydrazide (FcDH) via dynamic covalent linkages. The resulting copolymer self-assembled into nanoparticles (P(CA-DOX-Fc)-PEG-NPs) with high DOX and FcDH contents of 64.6 and 15.9%, favorable stability, and a minimal premature leakage of <1% in 160 h. Under acidic and oxidative conditions, the nanoparticles underwent self-amplified degradation, triggered by glutathione (GSH) depletion-induced ROS elevation and Fc-catalyzed hydroxyl radical (·OH) generation, enabling enhanced DOX release and ferroptosis induction. The dual-stimuli responsiveness ensured selective activation in the TME, and cellular uptake studies confirmed effective internalization and nuclei accumulation of DOX in HepG2 cells. In vitro cytotoxicity assays showed a low half-maximal inhibitory concentration (IC) of 8.91 μg/mL for the HepG2 cells, high viability in normal L02 cells, and a combination index (CI) of 0.92, indicating synergistic chemo- and ferroptosis therapeutic effects. These results demonstrate that P(CA-DOX-Fc)-PEG-NPs offer a promising strategy for precise, tumor-specific, and self-amplified combination therapy through ROS replenishment and environmentally triggered drug release.

Aminoglycosides (AGs) are among the earliest known classes of antibiotics. Despite decades of clinical utility, they have become largely ineffective due to the spread of antimicrobial resistance. In an effort to improve the activity of AGs against resistant strains, we conjugated them with antisense oligonucleotides, specifically peptide nucleic acids (PNAs). We report the synthesis and biological evaluation of novel neomycin (NEO) and amikacin (AMK) conjugates with 10-mer PNA oligomers targeting the essential bacterial gene encoding the acyl carrier protein. The conjugates were prepared via copper(I)-catalyzed azide-alkyne cycloaddition between 5″-azido-modified NEO or 6″-azido-modified AMK and alkyne-functionalized PNA. The AG-PNA conjugates exhibited higher antibacterial activity against AG-resistant strains than the parent AGs or mixtures of unconjugated components, with the NEO-PNA conjugate showing activity against NEO-resistant Typhimurium LT2 at an 8 μM concentration. Experiments using mismatched-sequence conjugates and conjugates with PNA sequences targeting the gene encoding red fluorescent protein confirmed the antisense mechanism's contribution to antibacterial activity. Membrane permeabilization assays demonstrated that PNA conjugation preserves AG interaction with the bacterial outer membrane, but alterations of the inner membrane potential and dependence on the SbmA transporter indicate the inner membrane as the main obstacle to bacterial uptake. These AG-PNA conjugates represent a promising strategy for overcoming AG resistance via a dual-action mechanism combining membrane interaction and antisense activity.

Multiple sclerosis (MS) is an autoimmune disease that causes neural degeneration as a result of the immune system launching an attack on the myelin sheath surrounding neurons. MS has multiple disease states; each one has been associated with a different onset pathway and requires a separate treatment. Primary progressive MS (PPMS) is a rare form of MS that affects 10-15% of MS patients, and Ocrelizumab is currently the only FDA-approved treatment on the market. While it can be effective in managing PPMS, Ocrelizumab can only delay the onset of the disease. In this study, MOG-Fc-BPI was designed as a potential therapeutic agent to suppress experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner, altering immune cells from an inflammatory to a regulatory phenotype. Here, MOG-Fc-BPI was successfully synthesized by conjugating the MOG-R peptide using sortase A enzyme to the C-terminus of the Fc-domain with LABL peptide at the N-terminus. Purified MOG-Fc-BPI was formulated to reach a concentration of 15 mg/mL for the in vivo study. MOG-stimulated EAE in C57BL/6 mice (a model for PPMS) that were treated with MOG-Fc-BPI on days 4 and 7 at 35 nmol/dose showed complete disease suppression on day 19 (score = 0; without symptoms) compared to PBS. The MOG-Fc-BPI-treated mice showed increased body weights throughout the study, while PBS-treated mice lost around 10% bodyweight during the peak of the disease without recovery up to the end of the study. Overall, this study provided a proof-of-concept that MOG-Fc-BPI has the potential to suppress PPMS.

In an attempt to broaden the scope of functional nucleic acids, phosphorescent platinum(II) complexes, resembling artificial metal-containing nucleobases, were attached covalently to DNA oligonucleotides via a deoxyribose moiety. The distance between the deoxyribose and the complex was varied by selecting three different linkers (propylene, ethylene, and methylene). Stable duplexes were obtained with any of the canonical nucleobases in the complementary position. When guanine was placed in this position, the most stable duplexes were obtained. No clear correlation was found between the identity of the linker and duplex stability. When two platinum(II) complexes were placed in adjacent positions within an oligonucleotide strand, photoluminescence spectra exhibited an additional broad low-energy band due to luminescence with excimeric character, indicating Pt···Pt interactions. The ratio of monomeric and excimeric emissions depends on the linker length and, interestingly, on the presence of dioxygen. Hence, a platinated oligonucleotide was developed into a ratiometric dioxygen sensor, capable of rapidly detecting dioxygen levels in volumes as small as 2 μL. The oligonucleotide proved to be nontoxic at relevant concentrations and could be transfected into cells, where it appeared to degrade so that further modification will be necessary to obtain an oligonucleotide-based ratiometric dioxygen sensor for intracellular measurements.

Glucagon-like peptide-1 receptor (GLP-1R) is overexpressed in >90% of insulinomas, making it an optimal target for imaging. However, current GLP-1R agonist tracers may induce side effects including hypoglycemia and nausea, particularly in pediatric patients. In this study, we employed a rational design approach combining molecular dynamics (MD) simulations with experimental validation to develop three Ga-labeled NOTA-conjugated exendin(9-39) derivatives featuring antagonist activity for safer imaging. MD simulations predicted differential binding affinities based on conjugation sites at Asp (E09), Lys (E12), and Lys (E27), with MM/GBSA calculations ranking E09 (-216.06 kcal/mol) > E12 (-200.01 kcal/mol) > E27 (-117.08 kcal/mol). Experimental validation through surface plasmon resonance confirmed these computational predictions, showing binding affinities consistent with the computational predictions. All radiotracers achieved radiochemical yields (>95%) and plasma stability (>91% intact after 120 min). In vivo PET imaging validated the computational hierarchy, with [Ga]Ga-E09 demonstrating superior tumor uptake (SUV: 3.99 at 60 min) compared with E12 (SUV: 0.75 at 60 min) or E27 (undetectable). These findings highlight the power of combining computational screening with systematic experimental validation. In conclusion, [Ga]Ga-E09 demonstrates superior binding affinity, cellular uptake, and imaging performance, suggesting its potential as a promising agent warranting further studies.

An affinity-guided photo-cross-linking reaction based on Fc-binding peptide harboring p-benzoyl-l-phenylalanine (PEptide-DIrected Photo-cross-linking; PEDIP) enables site-specific modification of native antibodies but suffers from issues coming from long UV exposure and high peptide concentrations. In this study, we report a bacterial surface-display system of the photo-cross-linking peptide and high-throughput screening of its libraries with FACS for higher photo-cross-linking efficiency. The lead peptide (B1) exhibited a higher conjugation yield than the original peptide (95.5% vs 78.4%) while preserving site fidelity at heavy chain Met252, confirmed by LC-MS/MS. A cyclooctyne group introduced to the N-terminus of B1 enabled conjugation of IgG with payloads via strain-promoted azide-alkyne cycloaddition. The conjugate of trastuzumab (antihuman HER2 IgG) and monomethyl auristatin retained antigen selectivity and exhibited potent cytotoxicity in HER2 HCC1954 cells with minimal activity in HER2 MDA-MB-231 cells.

Breast cancer remains a leading cause of cancer-related death worldwide, partly due to disease heterogeneity and the lack of reliable biomarkers. The G protein-coupled oxytocin receptor (OTR) has emerged as a potential biomarker and therapeutic target in breast cancer, as its overexpression correlates with tumor growth and metastasis. OTR thus presents new opportunities for molecular imaging and targeted therapy in breast cancer. This study explores three novel Ga-labeled peptides as potential OTR-specific imaging agents. Their preclinical evaluation includes in vitro assays and positron emission tomography (PET) studies in breast cancer models. The work also introduces the application of linchpin chemistry with and as a novel strategy for attaching bifunctional chelating agents. This tandem approach not only enables efficient peptide cyclization but also facilitates radiometal incorporation, representing a versatile platform for the design of next-generation radiopharmaceuticals. Binding studies using an aequorin-based assay in CHO cells expressing human OTR revealed the following EC values: (376 nM), (1.38 nM), and (123 nM). Radiolabeling with Ga was efficient and reproducible, consistently yielding high decay-corrected radiochemical yields of 52-74% and high radiochemical purity >98%. PET imaging demonstrated maximum MCF-7 tumor uptake for (SUV 0.64 ± 0.10; = 3) and (SUV 0.64 ± 0.05; = 7) at 10 min postinjection, whereas reached comparable uptake (SUV 0.64 ± 0.12; = 3) at 30 min. Notably, showed superior background clearance and faster blood pool washout. Tumor uptake specificity was verified through competitive inhibition studies: predosing with oxytocin reduced tracer accumulation in a concentration-dependent manner at 10 min postinjection, with decreases of 33% at 50 μM and 68% at 300 μM, confirming selective OTR-mediated binding in vivo. Among the evaluated tracers, the novel peptide demonstrated efficient radiolabeling, strong binding potency, and favorable in vivo characteristics, including uptake in estrogen receptor-positive MCF-7 tumors and superior background and clearance profiles. With further structural optimization, holds promise as a PET radioligand for targeting OTR in breast cancer.

Therapeutic mRNA has received significant attention as a new class of nucleic acid-based medicine due to its promising potential toward protein replacement therapy, vaccine development, and genome editing. Unlike DNA-based therapies, which depend on nuclear entry, mRNA works directly in the cytoplasm to produce proteins. Nevertheless, the delivery of large nucleic acids, such as mRNA, remains an unresolved challenge due to their instability, limited cellular uptake, and the cytotoxicity commonly associated with several cationic carriers. In recent years, various platforms have been developed for delivering mRNAs, including lipid nanoparticles, liposomes, dendrimers, and polyion complex micelles. Despite their success, each of these platforms faces important challenges, such as cytotoxicity, poor encapsulation efficiency and stability, limited endosomal escape, and reduced effectiveness in biological media. As a general alternative, in this study, we developed a peptide-based artificial viral capsid modified with a cell-penetrating peptide possessing perfluoroalkyl (PFA) chain (CADF) for the efficient and safe delivery of mRNAs into cells. The PFA-modified artificial viral capsid was formed by the self-assembly of the CADF-conjugated β-annulus peptide, unmodified β-annulus, and dT-SS-β-annulus, which can be hybridized with the poly(A) tail of the mRNA. Dynamic light scattering and transmission electron microscopy confirmed the formation of mRNA-encapsulated spherical capsids of approximately 200 nm in diameter. Importantly, PFA modification of the artificial viral capsid significantly improved the delivery efficiency and minimized cytotoxic effects. Fluorescence images further demonstrated that cells treated with these capsids exhibited significantly higher expression of mCherry-encoding mRNAs, indicating successful delivery and translation. Overall, our study introduces a promising viral-mimetic approach for mRNA therapeutics without compromising safety and efficiency.

Red blood cells (RBCs) have been employed to convey and deliver a variety of therapeutic agents, from small molecules to proteins. The therapeutics are typically installed within the RBC interior via a pore-forming process that results in membrane disruption and a partial loss of hemoglobin. An alternative approach, namely appending therapeutics to the RBC surface, has received significantly less attention. Here we focus on the characterization of an array of membrane anchoring modalities (noncovalent, reversible covalent, and covalent). Surface modification is experimentally simpler and structurally less invasive than its membrane disruptive counterpart. This panel is designed, synthesized and assessed with respect to RBC loading capacity, retention, and rate of transfer to other cell populations. The cell surface anchors are appended to a structural scaffold (cobalamin) that can house and deliver therapeutic agents. Imaging studies for a series of representative derivatives reveal that these species are not internalized by the RBCs, consistent with the absence of an active endocytic pathway in mature RBCs. Furthermore, enzymatic digestion of the glycocalyx failed to impair loading or retention, suggesting that the derivatives are likely anchored to the RBC membrane. The structural motifs identified in this study provide a template for the development of membrane tethered therapeutics that are specifically designed to be transported to diseased sites by RBCs.

The scarcity of a simple, cost-effective, and green method for the immobilization of enzymes severely hampers their application. Herein, a versatile and mild xylanase and lichenase bienzyme (XLBE) immobilization strategy including biofunctionalization of the magnetic particles, enzyme-free purification, and spontaneous covalent bridging based on SpyCatcher "Click Biology" was proposed. Only biocompatible tannic acid (TA), Fe, and elastin-like polypeptide-SpyCatcher were fed. The biomodified magnetic particles exhibited excellent stability with a loss of only 3.51% EC after 1 h of incubation at pH 7.5. Then, they were applied to immobilize SpyTag fused XLBE directly from the crude solution at a loading of 12.5 mg/g. The retention of XLBE and the xylanase activity were as high as 87.73% and 82.77%, respectively. The half-lives of the immobilized xylanase increased by 1535.75% (50 °C) compared to those of the free xylanase. The immobilized XLBE showed excellent reusability, retaining 70.15% (xylanase) and 78.81% (lichenase) of the initial activity after 8 cycles of recycling. They also showed superior catalytic performance with 202.25% improvement in green production of total reducing sugar and 30.77% improvement in juice clarification. Moreover, the versatility of the immobilization strategy was also demonstrated on inorganic carriers such as silicon dioxide and carbon nanotubes. This innovative all-in-one strategy avoids too many chemical reagents for surface modification and omits the complex enzyme prepurification process for immobilization, which will shed light on the green biocatalytic applications based on time-effective and low-byproduct surface functionalization strategies.

T-cell immunoglobulin and mucin domain 3 (TIM-3), a critical immunosuppressive checkpoint receptor, regulates antitumor immunity within the tumor microenvironment (TME). Noninvasive quantification of TIM-3 expression could be helpful for guiding immunotherapy and monitoring treatment response. We developed Ga-DOTA-D-P24, a novel D-configured peptide radiotracer designed for enhanced protease resistance and optimized TIM-3 targeting. Radiolabeling yielded high radiochemical purity (RCP) (>98%) and excellent / stability. Positron emission tomography (PET)/CT imaging across six tumor models demonstrated that Ga-DOTA-D-P24 showed high specific uptake in MGC-803 gastric carcinoma. Comparative PET studies showed that the D-configured tracer exhibited 1.6-fold higher tumor uptake than that of Ga-DOTA-L-P24. Furthermore, Ga-DOTA-D-P24 successfully visualized the interleukin-15 (IL-15)-triggered elevation of TIM-3 expression in tumors, demonstrating its potential as a noninvasive tool for assessing target engagement and treatment response in TIM-3-associated malignancies.

Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling the modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargo to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent antileukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables the customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.

Precise glioma detection is a critical challenge in the clinic. Magnetic particle imaging (MPI) is an emerging, highly sensitive medical imaging technique that has the potential to accurately detect glioma at the molecular and cellular levels. Magnetic nanoparticles (MNPs) provide an effective approach for targeted imaging to specific regions, and the morphology of MNPs plays a vital role in determining their MPI performance. MNPs with various shapes have been developed to pursue sensitive MPI, while the effect of the multivoid structure on MPI tracers is still unrevealed. Herein, we systematically investigate the impact of multivoid, yolk-shell, and completely hollow structures on the MPI signal. We identify that an increased number of magnetic cores per unit volume, decreased coercivity, and reduced full width at half-maximum of the magnetization derivative caused by the multivoid structure are the key factors that endow tracers with high MPI sensitivity. Moreover, further Arginine-Glycine-Aspartic Acid peptide modification ensures that the multivoid nanotracer exhibits high affinity and targeting to tumor cells and tissues, providing an obvious MPI signal to achieve precise glioma detection. This work enables a fundamental understanding of the effect of the multivoid structure on the MPI signal, lending guidance for designing high-performance MPI tracers for biomedical applications and promoting precise disease diagnosis.

Bicyclic peptides, with two cyclic substructures, have emerged as a powerful tool for modulating challenging targets such as protein-protein interactions. Meanwhile, DNA-encoded library technology (DELT) provides a powerful platform for hit discovery. The unity of both fields has the potential to identify potent bicyclic ligands for the targets of interest. Therefore, there is a high demand to develop an efficient way to construct bicyclic peptide libraries. Herein, we describe a novel and efficient approach to the synthesis of DNA-encoded bicyclic peptides via a cysteine-promoted cyclization and amide condensation reaction. This strategy proceeds smoothly under mild conditions and can generate a wide range of bicyclic peptides with various peptide sequences and ring sizes in good conversions.

DNA topoisomerase I (TOP1) inhibitor-based antibody-drug conjugates (ADCs) incorporating photosensitive camptothecin (CPT) analogs as payloads have emerged as a promising therapeutic strategy in oncology. However, their clinical potential is challenged by photoinduced instability during manufacturing, storage, and handling, which are typically conducted under ambient light conditions, using white light with wavelengths greater than 400 nm and minimal ultraviolet (UV) exposure. In this study, we systematically investigated, for the first time, the impact of ambient light exposure on TOP1 inhibitor-conjugated ADCs (TOP1-ADCs), and we revealed critical photodegradation mechanisms that compromise their physicochemical properties and therapeutic efficacy. Upon ambient light exposure, TOP1-ADCs underwent significant chemical, physical, and biofunctional changes, including visible color changes, aggregation, oxidation, drug loss, payload degradation, destabilization in C2 domain, and reduced binding affinity to the neonatal Fc receptor (FcRn). Mechanistic studies revealed two distinct pathways driving this photodegradation: a reactive oxygen species (ROS) generation-mediated pathway and a direct payload self-photolysis-mediated pathway. In oxygen-rich environments, the ROS-generation-mediated pathway predominates, where the excited-state payload primarily transfers energy to molecule oxygen to induce ROS formation, leading to oxidation and subsequent aggregation and drug loss. Under oxygen-depleted conditions, direct payload photolysis becomes the primary degradation mechanism, resulting in payload degradation and more severe particular nonreducible aggregation formation. These findings highlighted the necessity of implementing stringent light-protective measures throughout the production, storage, and handling of TOP1-ADCs to preserve their stability, efficacy, and safety. The study provided critical insights into the photosensitivity of TOP1 inhibitor-based ADCs, offering a foundation for optimizing their development and clinical applications.

Cutaneous melanoma, responsible for 80% of skin cancer mortality, presents urgent diagnostic challenges due to insufficient early detection methods. Current clinical methods rely on invasive biopsies, while noninvasive approaches primarily serve as adjunctive decision-support tools rather than definitive diagnostics. Here, a peptide-based fluorescent biosensing system was developed for the sensitive and rapid detection of S100B, a key prognostic biomarker for melanoma. Our system employs a fluorescently labeled peptide beacon designed for Förster resonance energy transfer (FRET)-based detection, achieving a subnanomolar detection limit (∼0.045 nM) and great selectivity in human serum samples. Peptide synthesis was performed using optimized solid-phase protocols, enabling precise sequence assembly, while the peptide sensor offers efficient detection, lower costs, and high specificity through tailored peptide-protein interactions. The biosensing probe employs complementary peptide nucleic acid (PNA) interactions to achieve proximity-induced fluorescence quenching in the absence of S100B, which reverses via structural rearrangement upon specific S100B binding for accurate quantification. Computational and experimental optimization of the synthetic process has enhanced binding efficiency, sensitivity, and response time-crucial parameters for melanoma-specific detection. By integrating advanced molecular design with optical biosensing, this mechanism aims to enhance the accuracy and accessibility of melanoma diagnostics, ultimately addressing healthcare disparities and improving patient outcomes.

E-selectin is a highly glycosylated protein overexpressed on the surface of endothelial cells within the tumor vasculature, especially at sites of active angiogenesis and metastasis. This localized overexpression raises the opportunity to target the tumor microenvironment by using nanocarriers capable of specific recognition by this protein. In this work, we report dual-peptide PAMAM dendrimer conjugates as novel nanocarriers with specific E-selectin-mediated uptake properties. The conjugates were obtained from fourth-generation PAMAM dendrimers that were partially acetylated and doubly conjugated with an E-selectin targeting peptide (CIELLQAR or CIELFQAR) and the cell-penetrating peptide pTAT. Acetylation degrees close to 50% were successfully achieved, with peptide substitution ratios corresponding to 3-4 pTAT units and 2-3 E-selectin targeting peptides per dendrimer, as determined from NMR analysis. The dual-peptide conjugated dendrimers showed excellent safety profiles, with negligible intrinsic cytotoxicity in HUVEC/TERT2 and T98G cells, as models for endothelial and tumor cells. Their E-selectin-mediated uptake was confirmed in endothelial cells overexpressing E-selectin and blocked in cells treated with an anti-E-selectin antibody, with the pTAT-CIELFQAR-conjugated dendrimer having a superior performance. The best dual-peptide dendrimer conjugate was also internalized by T98G cells, which was attributed to the pTAT cell internalization properties. In preliminary assays, this system proved capable of delivering doxorubicin to tumor cells, which highlights the potential of this functional dendrimer conjugate as a novel platform for targeted cancer therapy.