Bioconjugate Chemistry最新文献

Chemo-photothermal therapy has emerged as a promising approach for clinical tumor treatment, effectively circumventing drug resistance and minimizing the side effects associated with chemotherapy (CHT) drugs. However, this therapeutic strategy is significantly limited by the poor accumulation capacity of the nanomaterials. In pursuit of innovative nanomaterials for precise diagnosis and effective treatment, an active targeted nanoplatform AuNR/Apt-M@DOX was constructed. This nanoplatform was fabricated by integrating gold nanorods (AuNR) with a polymorphic epithelial mucin (MUC1) aptamer (Apt-M). Concurrently, doxorubicin (DOX) was intercalated into the DNA double helix formed by Apt-M, enabling targeted drug delivery. Facilitated by Apt-M-mediated recognition and endocytosis, the nanoplatform was selectively delivered into the tumor site. Subsequently, the excellent photothermal properties of AuNR under NIR irradiation enabled effective photothermal therapy (PTT). Meanwhile, a weak acidic environment and NIR laser simultaneously triggered the release of DOX, achieving chemotherapy (CHT). Under NIR laser irradiation, the nanoplatform exhibited excellent performance in chemo-photothermal therapy and successfully inhibited tumor growth. This work presents a novel concept of a nanoplatform for enhanced tumor ablation, dual stimuli-responsive drug release, and the combination of CHT and PTT in a drug-loaded nanoplatform.

Cell-surface conjugation has enormous therapeutic and research potential. Existing technologies for cell-surface modification are usually reversible, nonspecific, or rely on genetic editing of target cells. Here, we present the NanoBondy, a nanobody modified for covalent ligation to an unmodified protein target at the cell surface. The NanoBondy utilizes the 20 naturally occurring amino acids, harnessing NeissLock chemistry engineered from Neisseria meningitidis. We evaluated the binding and specificity of a panel of nanobodies to CD45, a long-lived surface marker of nucleated hematopoietic cells. We demonstrated the conversion of existing nanobodies to covalently reacting NanoBondies using a disulfide clamp to position the self-processing module of FrpA close to the nanobody antigen-binding site. The addition of calcium induces anhydride formation at the NanoBondy C-terminus, enabling proximity-directed ligation to surface amines on CD45. We optimized the NanoBondy reaction by fine-tuning linkers and disulfide clamp sites to modulate anhydride positioning. Tandem mass spectrometry mapped reaction sites between NanoBondy and CD45. NanoBondy ligation was robust to buffer, pH, and temperature and was detected within 2 minutes. We established the reaction specificity of NanoBondies to endogenous CD45 at the surface of NK cells and T cells. NanoBondy technology provides a modular approach for targeted, inducible, and covalent cell-surface modification of immune cells without their genetic modification.

Despite the powerful antitumor activity of amonafide (ANF), its use in the clinic has been limited by dose-dependent toxicities. To address this challenge, we developed a series of unsaturated fatty acid-amonafide conjugates, leveraging the biocompatibility and inherent tumor-targeting capacity of unsaturated fatty acids. Through amidation reactions, five conjugates were synthesized and evaluated. ANF-DHA stood out as the most promising candidate, demonstrating not only targeted cytotoxicity against cancer cells but also the ability to overcome chemoresistance. Additionally, the design allows for selective fluorescence activation by fatty acid amide hydrolase (FAAH), making it useful for monitoring ANF release in cancer cells. Mechanistic studies revealed that ANF-DHA induces autophagic cell death, effectively disrupting survival pathways in drug-resistant tumors. Our results indicate that modifying ANF by conjugating it with unsaturated fatty acids represents a promising therapeutic platform for enhancing tumor targeting, mitigating side effects, and enabling real-time visualization of drug release.

Bioconjugates have increasing utility in numerous medical and materials applications; thus, the development of new mechanisms to increase their valency and functional potential has the ability to further their impact. Expansion of the chemical tools used to prepare bioconjugates affords greater flexibility in their preparation and can improve their potency and specificity. This research integrates genetic code expansion methodologies with bioorthogonal reaction development to prepare homogeneous multivalent bioconjugates. Specifically, a novel bioorthogonal reaction has been optimized, reacting an O-alkoxylamine with a 1,3-diyne in the absence of any additional reagents. This reaction has been found to progress to near completion in under 30 min and generate highly stable bioconjugates. Utilizing a cascade sequence involving a bioorthogonal Glaser-Hay coupling, followed by treatment with an aminooxy partner, provides a mechanism to introduce two novel functionalities into proteins. Moreover, the precise control of genetically incorporating an alkynyl amino acid at a specific residue provides a high degree of control over the conjugate structure and activity. This cascade reaction was also optimized to occur in a one-pot fashion, obviating the need for conjugate purification between reactions. Finally, this strategy was employed in producing a highly effective antibody-drug conjugate (ADC) functionalized with monomethyl auristatin E (MMAE) and a fluorescent probe, allowing for monitoring of therapeutic delivery. When tested against HER2+ cells, this trivalent conjugate was specific, potent, and trackable. As this simple proof-of-concept demonstrates, there is limitless potential for the preparation of other therapeutic and diagnostic bioconjugates using this novel approach.

Synthetic multimeric peptide ligands of the natural cytotoxicity receptor, NKp30, on natural killer cells were developed in this study. A divergent solid phase peptide synthesis strategy was optimized for the conjugation of multiple peptide ligands, based on the parent TVPLN and related permutation sequences, into branched and hyperbranched peptides for structure-activity relationship studies. According to CD spectroscopy in aqueous trifluoroethanol, the parent TVPLN peptide transitioned from a β-turn (monomer and dimer) to an extended β-sheet and a helix-type conformation in its branched (trimer) and hyperbranched (tetramer) structures. The multimeric peptides were predicted to expand the binding interface to NKp30 according to molecular modeling and docking predictions. Flow cytometry revealed greater binding activity of the trimer and tetramer ligands onto NKp30-coated beads and the natural killer cells, while being displaced by the native B7H6 ligand in competitive binding studies on the NKp30-coated beads and reducing anti-NKp30 binding on the natural killer cells, suggesting some receptor-specific binding activity. Immunostimulatory activity assays showed little secretion of TNF-α and IFN-γ from the natural killer cells following peptide treatment, with the TVPLN monomer and dimer remaining superior to the rest. Select changes in sequence compositions and the ligated multimeric peptide display had an inhibitory effect on natural killer cell activation. This result was likely due to changes in bound vs unbound multimeric peptide structures that deviated from the bioactive β-turn of the TVPLN monomer and dimer for direct peptide engagement at the NKp30 active site. Nonetheless, the novel multimeric peptides showed improved cell binding activity relative to their linear counterparts and were nontoxic at lower (10 μM) doses, making them safe and effective for structure-activity studies for the discovery of novel peptide ligands of natural killer cells.

Zelenectide pevedotin is a Nectin-4 targeting Bicycle drug conjugate® (BDC®) that has fast track designation for the treatment of bladder, breast, and lung cancers. Its structure is highly complex and comprises a Bicycle connected to microtubule inhibitor monomethyl aurostatin-E (MMAE) via a molecular spacer and a cleavable linker. This provides a unique challenge in structural elucidation, and this report shows a strategy to overcome this using an incremental, nuclear magnetic resonance spectroscopy-based approach, which may be applied to complex structures of a similar nature.

Gas vesicles (GVs) are air-filled protein nanoparticles that are proving to be useful in a number of biomedical applications. We hypothesized that it could be possible to develop a modular method for creating rapidly prototyped GVs by modifying their surface chemistry to include targeting peptides in an orientation-specific manner. Here, we describe a modular method to create targeted GVs using His-tagged antibody fragments, ensuring that the antibody fragments are connected to the GV in an orientation-specific manner. This is achieved via the functionalization of the GVs with the nickel-nitrilotriacetic acid (Ni-NTA) group. First, we validated that these functionalized GVs can bind His-tagged green fluorescent protein and characterized the particle size and surface charge of functionalized GVs. Then, GVs targeted to prostate-specific membrane antigen (PSMA) using a minibody were validated using a knockout validation in vitro.

The tetrazine ligation, an inverse electron-demand Diels-Alder reaction between tetrazine and a dienophile such as norbornene (Nb) or trans-cyclooctene (TCO), has characteristics that make it a potentially ideal chemistry for the preparation of nanoparticle bioconjugates. Although this click chemistry has been used for this purpose, relatively little is known about how to optimize nanoparticle surface chemistry and tagging with reactive functional groups to maximize the chances of successful conjugation. Here, we addressed this open question with quantum dots (QDs) by preparing and testing a panel of coatings for tetrazine ligation with a small-molecule fluorescent dye and an immunoglobulin G antibody. These coatings included Nb-appended dithiol-anchored ligands based on a small-molecule design, a poly(ethylene glycol) (PEG) oligomer, and multiple dextran variants. Additional coatings included a PEGylated amphiphilic polymer and a PEGylated dithiol-anchored coordinating polymer, both of which were modified with TCO, Nb, or tetrazine groups. The Nb-appended ligands lost their reactivity between bulk solution and binding to the QDs, whereas both types of polymer-coated QD were reactive toward a small-molecule fluorescent dye, regardless of the click-reactive group tagged on the PEG. However, the efficiency of the ligation of both polymer coatings with antibodies was highly dependent on whether the dienophile was Nb or TCO, whether this group was tagged on the QDs or the antibodies, and if the polymer was amphiphilic or coordinating. Immunofluorescent labeling of a cancer cell line revealed that the greater versatility of the coordinating polymer coating for ligation came at the expense of substantial nonspecific binding to cells, which was avoided by the amphiphilic polymer coating. These observations and trends are discussed to arrive at recommendations for maximizing the probability of successful tetrazine ligation to form bioconjugates from QDs and other colloidal nanoparticles.

The blood-brain barrier (BBB) remains the single most obstructive bottleneck to mRNA therapeutics for central nervous system (CNS) disorders. Here we introduce a metabolically primed, redox-locked nanoplatform that converts the brain's glucose addiction into a gate-cracking key. A disulfide-stabilized, glucose-decorated block-cationic copolymer self-assembles with mRNA into 36 nm micelles (ζ potential approximately +4.5 mV) that withstand serum nucleases and polyanionic assault yet disassemble on cytosolic glutathione. Fasting-induced GLUT1 upregulation (4.2-fold) is exploited as a transiently overexpressed "receptor"; a subsequent glycemic spike drives receptor-mediated transcytosis, yielding 160-fold higher cerebral mRNA accumulation versus untargeted controls. Single-cell intravital imaging confirms parenchymal penetration within 120 min and pan-brain GFP expression. The strategy affords spatiotemporally sharp CNS transfection without BBB disruption or systemic toxicity, offering a generalizable, nonviral avenue for genomic medicine of neurological diseases.

Modular, tunable linker chemistries that are stable in circulation yet selectively cleaved are needed to realize the therapeutic potential of antibody-drug conjugates (ADCs). Near-infrared (NIR) fluorogenic imaging using norcyanine carbamate (CyBam) probes can quantitatively compare ADC linkers across in vitro and in vivo settings. A series of substituted CyBams modified with phosphoramidates, peptides, and control triggers were conjugated to EGFR-targeting monoclonal antibody (mAb) panitumumab. Imaging in cellular and in vivo settings reveals the potential for novel phosphoramidate linkers, showing selective cellular activation, excellent tumor localization, and reduced liver signal compared to conventional proteolytic linkers. Guided by these imaging results, monomethyl auristatin E (MMAE) conjugates bearing the new linkers were prepared and displayed picomolar potency, receptor-dependent activity, and promising in vivo tumor growth inhibition. In total, these studies demonstrate that quantitative fluorogenic imaging can enable the discovery and prioritization of new ADC linkers.