Bioconjugate Chemistry最新文献

Therapeutic antibodies have transformed the treatment of cancer, inflammation, infection, and disorders of bone biology by recognizing disease-associated molecular targets with high specificity. However, achieving therapeutically effective antibody concentrations at the site of disease remains a major challenge, as systemic administration does not inherently result in efficient tissue-specific delivery. Bone is a particularly demanding destination because mineralized matrix, limited vascular access, marrow complexity, and active remodeling can restrict local antibody exposure. Recent advances in bone-targeting antibody design point to a powerful reframing in which antibodies can be engineered not only for antigen specificity but also for tissue specificity. By attaching hydroxyapatite-binding motifs such as bisphosphonates or acidic peptides, antibodies can be endowed with bone-homing capabilities. The efficacy of this strategy has now been validated across multiple preclinical disease models, including breast cancer bone metastasis and osteoporosis-related bone loss. The next challenge is to define how affinity, valency, conjugation site, circulation time, and release should be tuned for each skeletal disease.

A polyacrylamide derivative of boric acid has been proposed for staining the cellular glycocalyx. Probes differing in their boric acid residue content and type of fluorescent label were obtained by conjugating fully activated polyacrylic acid with aminophenylboronic acid and amino derivatives of dyes. Maximal glycocalyx staining and, at the same time, minimal penetration inside the living cell were observed with a ∼20 kDa probe containing fluorescein and phenylboronic acid residues at a loading of 30 mol %. The patch-like staining reaches its maximum within a few minutes, is irreversible (the probe does not wash out of the glycocalyx), and partly differs from lectin staining.

Protein filaments, including silk, collagen, and amyloids, are natural building blocks whose hierarchical self-assembly produces adaptive mechanics and diverse functions. Conventional protein engineering has had limited success in resolving sequence-structure-function relationships across length scales. Recent advances in artificial intelligence (AI) now provide a framework for physics-informed multiscale design, from molecular motifs to functional macroscopic architectures. Machine-learning models can encode biophysical principles and guide the generation of protein-based materials. When combined with high-throughput experimentation, generative AI can integrate sequence design, structure prediction, and functional validation into closed-loop workflows. These workflows can accelerate the development of programmable biomaterials. This review summarizes AI-driven advances in protein-based materials and emerging links with autonomous robotics.

Many clinically used chemotherapeutics, including doxorubicin, suffer from dose-limiting toxicities, poor biodistribution, and limited control over intracellular activation. To mitigate these challenges, two primary delivery strategies have been widely explored: prodrug design and liposomal encapsulation. Prodrugs can improve solubility, pharmacokinetics, and site-selective activation, while liposomes enhance circulation time and reduce off-target exposure. However, each approach presents limitations when employed independently. Prodrugs may introduce stability or solubility concerns and can undergo premature activation, whereas liposomal formulations often rely on passive release mechanisms and may exhibit incomplete or non-specific cargo leakage. In the current work, we sought to develop a synergistic delivery approach that integrates a chemically responsive prodrug directly within the liposomal membrane that responds to reactive oxygen species (ROS) highly upregulated within diseased cells. We report a ROS-responsive doxorubicin lipid prodrug conjugate, ROSDoxL, which is incorporated within the liposomal bilayer, enabling membrane-embedded therapeutic loading rather than aqueous core encapsulation. The prodrug design features a boronate caging group that undergoes oxidation in the presence of elevated reactive oxygen species, triggering a self-immolative release of doxorubicin. A non-responsive control doxorubicin lipid, DoxL, lacking the boronate moiety, was used to evaluate the specific ROS-mediated uncaging mechanism. ESI-HRMS studies confirmed ROS-dependent cleavage of ROSDoxL, while liposomal characterization and cellular studies demonstrated functional delivery and concentration-dependent cytotoxicity in A375 melanoma cells comparable to free doxorubicin. Our membrane-integrated liposomal prodrug approach provides a modular strategy for coupling disease-associated biochemical signals to controlled drug activation, advancing liposomal systems beyond passive carriers towards stimulus-responsive therapeutic delivery.

Trophoblast cell surface antigen 2 (TROP-2) is a transmembrane glycoprotein overexpressed across a range of epithelial malignancies, yet its clinical assessment still relies largely on invasive immunohistochemical analysis of biopsied tissues. In this study, we employed the PepMimic artificial intelligence platform to de novo design a TROP-2-targeting peptide, TR23, via binding interface mimicry, which was subsequently conjugated with DOTA and radiolabeled with ⁶⁸Ga to yield the peptide-based PET probe [⁶⁸Ga]Ga-DOTA-TR23. The tracer bound TROP-2 with nanomolar affinity (KD = 26.8 nM), showed selective cellular uptake in TROP-2-positive cells, and exhibited high radiochemical purity (>95%) with excellent stability in saline and serum. Micro-PET/CT imaging in pancreatic (BxPC-3), prostate (PC3), and thyroid (BCPAP) xenograft models revealed rapid, specific tumor accumulation with high contrast and low background uptake, while competitive blocking studies with excess unlabeled TR23 substantially suppressed tumor uptake, confirming receptor-mediated specificity. Quantitative analysis further demonstrated strong positive correlations between SUVmax and TROP-2 expression levels verified by immunohistochemistry across all three tumor types. Collectively, these findings establish [⁶⁸Ga]Ga-DOTA-TR23 as a promising peptide-based PET tracer for rapid, specific, and quantitative imaging of TROP-2-positive tumors, and highlight the PepMimic-enabled strategy as a versatile platform for peptide-based probe discovery, supporting the translational potential of this tracer as a pan-cancer diagnostic agent for precision molecular imaging.

This study presents a rational, simulation-guided strategy to overcome the structural instability of ferritin nanocages upon conjugation with large biomoleculesa common challenge in protein-based bioconjugates. Molecular dynamics (MD) simulations were performed using epidermal growth factor (EGF) and enhanced green fluorescent protein (eGFP) as models, which predicted that excessive eGFP fusion induces steric hindrance, disrupts hydrogen bonding, and weakens binding free energy at critical C3 interfaces, thereby destabilizing the cage. Based on these insights, an in vitro heteromeric assembly strategy was designed, employing EGF- and eGFP-(G₄S)₃-FTH1 subunits to generate pure, dual-functional nanostructures as a genetically programmable alternative to chemical bioconjugation. Successful assembly and functionality were confirmed by electron microscopy, fluorescence, and cell binding assays. To quantitatively assess stability, comprehensive biophysical profiling was performed under thermal, pH, and salt stress. Thermal denaturation, as monitored by nanoDSF, native PAGE, and qPCR-DSF, revealed a stepwise disassembly pathway: early local rearrangements at C3 interfaces precede global cage dissociation, and the disassembly midpoint temperature (T_diss) of the intact 24-mer increased from 62.9 °C to 79.2 °C as the EGF-FTH1 proportion increased, consistent with MD-predicted weakening of C3-interface hydrogen bonds and binding energy. Although a proportion of 40% EGF-FTH1 subunits (with 60% eGFP-(G₄S)₃-FTH1) represented the minimal threshold for forming intact heteromeric cages, 60% EGF-FTH1 (with 40% eGFP-(G₄S)₃-FTH1) provided the optimal balance between functional display and stability, retaining >80% cage integrity at pH 7, exhibiting superior salt tolerance, and maintaining one-month storage stability at 4 °C; increasing the eGFP-(G₄S)₃-FTH1 ratio beyond 40% led to markedly reduced stability. The co-assembly approach was validated with TAT/EGF-functionalized cages, which maintained stability and enhanced cellular uptake. MD-predicted subunit interfaces were thus used to guide the combination of stable and unstable subunit pairs, yielding protein nanocages with balanced functionality and stability, and establishing this strategy as a versatile approach for expanding the utility of multifunctional protein nanocages in biomedicine and industrial biocatalysis.

Photoacoustic imaging (PAI) combines optical absorption with ultrasonic detection and has emerged as a powerful platform for noninvasive molecular imaging. The clinical value of a photoacoustic contrast agent (PACA) is not only determined by tumoral accumulation, but also by whether it can generate sufficient target-to-background contrast. In practice, PACAs can be designed to detect tumor-associated biomarkers in two complementary strategies. Molecularly targeted PACAs rely on specific biological recognition to enhance probe localization or retention, whereas an activatable probe generates photoacoustic signals only in response to a disease-related biochemical trigger. Recent studies further show that molecular targeting and activatable sensing can be integrated within a single probe, for example, by combining receptor recognition with activatable, ratiometric, or photo switchable signal generation. We argue that next-generation PACAs should move beyond maximizing tumor uptake toward maximizing biologically informative target-to-background contrast under clinically realistic imaging conditions.

Liposomal delivery has emerged as an effective way to transport a broad range of therapeutics due to the excellent biocompatibility of these nanocarriers and their capacity to encapsulate a wide variety of cargo. However, challenges still persist in controlled content release at diseased cells. Although several stimuli have been recently targeted to achieve effective liposomal triggered release at the target site, cysteine and glutathione (GSH), which are overabundant in cancer cells and play crucial roles by deterring oxidative stress and enhancing cell survival offer important targets. Here, we introduce a unique cysteine/GSH-responsive liposomal platform, which was achieved by incorporating a newly developed SLPC lipid containing a disulfide moiety inserted within the sn-2 acyl chain. Förster Resonance Energy Transfer (FRET)-based fluorescence assays, transmission electron microscopy, and dynamic light scattering studies confirmed the formation of stable spherical liposome vesicles containing our SLPC lipid switch and cysteine-responsive controlled cargo release. Selectivity screening with cysteine, GSH, homocysteine, dithiothreitol, methionine, serine, glutathione disulfide (GSSG), alanine, and glutamate demonstrated that SLPC-containing liposomes show a triggered response only in the presence of molecules containing active thiols. These findings demonstrate that SLPC can act as a potent liposomal platform to achieve cysteine/GSH-triggered controlled content release toward the goal of targeted drug delivery to diseased cells.

Aryl diazonium ions (ADIs) are well established for their reaction with both proteins and DNA, and yet these reactions have been studied independently without systematic comparison. When electrophilic bioconjugation probes are introduced to environments containing multiple macromolecules, questions of selectivity and competitive reactivity become critical. Here, we establish a systematic method to evaluate the selectivity of a series of ADIs toward proteins, specifically tyrosine and histidine residues, across varying pH and solvent conditions, while also looking at the ADI reaction with DNA. Electron-deficient ADIs preferentially modified tyrosine over histidine, but all tested ADIs reacted with and degraded DNA. We also observed strong pH- and hydrophobicity-dependent effects on amino acid selectivity. These findings provide a framework for understanding and controlling ADI reactivity in complex biological environments, informing the design of selective bioconjugation strategies.

We report the development of an injectable hyaluronic acid (HA)-based hydrogel using native chemical ligation (NCL) as a biocompatible and chemoselective crosslinking strategy for potential biomedical applications. High-molecular-weight HA was pre-modified with a glycinyl thioester derivative, while a bis-cysteine peptide served as the crosslinker, enabling rapid hydrogel formation under physiological conditions. After optimization of NCL parameters, the resulting hydrogel exhibits all key attributes for biomedical hydrogel injectables: cytocompatibility, injectability, tunable rheology adapted for soft-tissue delivery (e.g., skin and subcutaneous tissue), enzymatic degradability by hyaluronidase, adjustable swelling, and long-term shelf-life stability. It can be sterilized by autoclaving for injection purposes and also functions as a drug delivery matrix for localized, in situ release. This study presents a robust and scalable HA-based platform, available as either an injectable hydrogel or an in situ cross-linkable solution, with broad potential in drug delivery, regenerative medicine, and aesthetic applications.