Bioconjugate Chemistry Bioconjugate最新文献

Currently, there is no effective treatment for glioblastoma multiforme (GBM), the most frequent and malignant type of brain tumor. The blood-brain (tumor) barrier (BB(T)B), which is composed of tightly connected endothelial cells and pericytes (with partial vasculature collapse), hampers nanomedicine accumulation in tumor tissues. We aimed to explore the effect of nanomedicine size on passive targeting of GBM. A series of size-tunable poly(ethylene glycol) (PEG)-grafted copolymers (gPEGs) were constructed with hydrodynamic diameters of 8-30 nm. Biodistribution studies using orthotopic brain tumor-bearing mice revealed that gPEG brain tumor accumulation was maximized at 10 nm with ∼14 dose %/g of tumor, which was 19 times higher than that in the normal brain region and 4.2 times higher than that of 30-nm gPEG. Notably, 10-nm gPEG exhibited substantially higher brain tumor accumulation than 11-nm linear PEG owing to the prolonged blood circulation property of gPEGs, which is derived from a densely PEG-packed structure. 10 nm gPEG exhibited deeper penetration into the brain tumor tissue than the larger gPEGs did (>10 nm). This study demonstrates, for the first time, the great potential of a nanomedicine downsizing strategy for passive GBM targeting.

Conventional serum markers often fail to accurately detect cholestasis accompanying many liver diseases. Although elevation in serum bile acid (BA) levels sensitively reflects impaired hepatobiliary function, other factors altering BA pool size and enterohepatic circulation can affect these levels. To develop fluorescent probes for extracorporeal noninvasive hepatobiliary function assessment by real-time monitoring methods, 1,3-dipolar cycloaddition reactions were used to conjugate near-infrared (NIR) fluorochromes with azide-functionalized BA derivatives (BAD). The resulting compounds (NIRBADs) were chromatographically (FC and PTLC) purified (>95%) and characterized by fluorimetry, ¹H NMR, and HRMS using ESI ionization coupled to quadrupole TOF mass analysis. Transport studies using CHO cells stably expressing the BA carrier NTCP were performed by flow cytometry. Extracorporeal fluorescence was detected in anesthetized rats by high-resolution imaging analysis. Three NIRBADs were synthesized by conjugating alkynocyanine 718 with cholic acid (CA) at the COOH group via an ester (NIRBAD-1) or amide (NIRBAD-3) spacer, or at the 3α-position by a triazole link (NIRBAD-2). NIRBADs were efficiently taken up by cells expressing NTCP, which was inhibited by taurocholic acid (TCA). Following i.v. administration of NIRBAD-3 to rats, liver uptake and consequent release of NIR fluorescence could be extracorporeally monitored. This transient organ-specific handling contrasted with the absence of release to the intestine of alkynocyanine 718 and the lack of hepatotropism observed with other probes, such as indocyanine green. NIRBAD-3 administration did not alter serum biomarkers of hepatic and renal toxicity. NIRBADs can serve as probes to evaluate hepatobiliary function by noninvasive extracorporeal methods.

Fibroblast activation protein (FAP) has recently gained significant attention as a promising tumor biomarker for both diagnosis and therapeutic applications. A series of radiopharmaceuticals based on fibroblast activation protein inhibitors (FAPIs) have been developed and translated into the clinic. Though some of them such as radiolabeled FAPI-04 probes have achieved favorable in vivo imaging performance, further improvement is still highly desired for obtaining radiopharmaceuticals with a high theranostics potential. In this study, we innovatively designed an FAPI ligand SMIC-3002 by changing the core quinoline motif of FAPI-04 to the quinolinium scaffold. The engineered molecule was further radiolabeled with ⁶⁸Ga to generate a positron emission tomography (PET) probe, [⁶⁸Ga]Ga-SMIC-3002, which was then evaluated in vitro and in vivo. [⁶⁸Ga]Ga-SMIC-3002 demonstrated high in vitro stability, nanomolar affinity for FAP (8 nM for protein, 23 nM for U87MG cells), and specific uptake in FAP-expressing tumors, with a tumor/muscle ratio of 19.1 and a tumor uptake of 1.48 ± 0.03 ID/g% at 0.5 h in U87MG tumor-bearing mice. In summary, the quinolinium scaffold can be successfully used for the development of the FAP-targeted tracer. [⁶⁸Ga]Ga-SMIC-3002 not only shows high potential for clinical translation but also offers insights into designing a new generation of FAPI tracers.

The chemical synthesis of homogeneously ubiquitylated histones is a powerful approach to decipher histone ubiquitylation-dependent epigenetic regulation. Among the various methods, α-halogen ketone-mediated conjugation chemistry has recently been an attractive strategy to generate single-monoubiquitylated histones for biochemical and structural studies. Herein, we report the use of this strategy to prepare not only dual- and even triple-monoubiquitylated histones but also diubiquitin-modified histones. We were surprised to find that the synthetic efficiencies of multi-monoubiquitylated histones were comparable to those of single-monoubiquitylated ones, suggesting that this strategy is highly tolerant to the number of ubiquitin monomers installed onto histones. The facile generation of a series of single-, dual-, and triple-monoubiquitylated H3 proteins enabled us to evaluate the influence of ubiquitylation patterns on the binding of DNA methyltransferase 1 (DNMT1) to nucleosomes. Our study highlights the potential of site-specific conjugation chemistry to generate chemically defined histones for epigenetic studies.

Biosensors based on immobilized antibodies require molecular strategies that (i) couple the antibodies in a stable fashion while maintaining the conformation and functionality, (ii) give outward orientation of the paratope regions of the antibodies for good accessibility to analyte molecules in the biofluid, and (iii) surround the antibodies by antibiofouling molecules. Here, we demonstrate a method to achieve oriented coupling of antibodies to an antifouling poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) substrate, using glycan remodeling to create antibody-DNA conjugates. The coupling, orientation, and functionality of the antibodies were studied using two analysis methods with single-molecule resolution, namely single-molecule localization microscopy and continuous biosensing by particle motion. The biosensing functionality of the glycan-remodeled antibodies was demonstrated in a sandwich immunosensor for procalcitonin. The results show that glycan-remodeled antibodies enable oriented immobilization and biosensing functionality with low nonspecific binding on antifouling polymer substrates.

Membrane tension is an important physical parameter of describing cellular homeostasis, and it is widely used in the study of cellular processes involving membrane deformation and reorganization, such as cell migration, cell spreading, and cell division. Despite the importance of membrane tension, direct measurement remains difficult. In this work, we developed a ratiometric fluorescent probe sensitive to membrane tension by adjusting the carbon chain structure based on polarity-sensitive fluorophores. The probe is sensitive to changes in membrane tension after cells were subjected to physical or chemical stimuli, such as osmotic shock, lipid peroxidation, and mechanical stress. When the polarity of the plasma membrane increases (the green/red ratio decreases) and the membrane tension increases, the relative magnitude of the membrane tension can be quantitatively calculated by fluorescence ratio imaging. Thus, the probe proved to be an efficient and sensitive membrane tension probe.

Cationic polymers offer an alternative to viral vectors in nucleic acid delivery. However, the development of polymer vehicles capable of high transfection efficiency and minimal toxicity has remained elusive, and continued exploration of the vast design space is required. Traditional single polymer syntheses with large monomer bases are very time-intensive, limiting the speed at which new formulations are identified. In this work, we present an experimental method for the quick probing of the design space, utilizing a combinatorial set of 90 polymer blends, derived from 6 statistical copolymers, to deliver pDNA. This workflow facilitated rapid screening of polyplex compositions, successfully tailoring polyplex hydrophobicity, particle size, and payload binding affinity. This workflow identified blended polyplexes with high levels of transfection efficiency and cell viability relative to single copolymer controls and commercial JetPEI, indicating synergistic benefits from copolymer blending. Polyplex composition was coupled with biological outputs to guide the synthesis of single terpolymer vehicles, with high-performing polymers P10 and M20, providing superior transfection of HEK293T cells in serum-free and serum-containing media, respectively. Machine learning coupled with SHapley Additive exPlanations (SHAP) was used to identify polymer/polyplex attributes that most impact transfection efficiency, viability, and overall effective efficiency. Subsequent transfections on ARPE-19 and HDFn cells found that P10 and M20 were surpassed in performance by M10, contrasting with results in HEK293T cells. This cell type dependency reinforced the need to evaluate transfection conditions with multiple cell models to potentially identify moieties more beneficial to delivery in certain tissues. Overall, the workflow employed can be used to expedite the exploration of the polymer design space, bypassing extensive synthesis, and to develop improved polymer delivery vehicles more readily for nucleic acid therapies.

Cancer immunotherapy has yielded remarkable results across a variety of tumor types. Nevertheless, the complex and immunosuppressive microenvironment within solid tumors poses significant challenges to established therapies such as immune checkpoint blockade (ICB) and chimeric antigen receptor T-cell (CAR-T) therapy. Within the milieu, tumor-associated macrophages (TAMs) play a significant role by directly suppressing T-cell functionality and fostering an immunosuppressive environment. Effective regulation of TAMs is, therefore, crucial to enhancing the efficacy of immunotherapies. Various therapeutic strategies targeting TAM modulation have emerged, including blocking TAM recruitment, direct elimination, promoting repolarization toward the M1 phenotype, and enhancing phagocytic capacity against tumor cells. The recently introduced CAR macrophage (CAR-M) therapy opens new possibilities for macrophage-based immunotherapy. Compared with CAR-T, CAR-M may demonstrate superior targeting and infiltration capabilities toward solid tumors. This review predominantly delves into the origin and development process of TAMs, their role in promoting tumor growth, and provides a comprehensive overview of immunotherapies targeting TAMs. It underscores the significance of regulating TAMs in bolstering antitumor therapies while discussing the potential and challenges of developing TAMs as targets for immunotherapy.

Development of bioconjugation strategies to efficiently modify biomolecules is of key importance for fundamental and translational scientific studies. Cysteine S-arylation is an approach which is becoming more popular due to generally rapid kinetics and high chemoselectivity, as well as the strong covalently bonded S-aryl linkage created in these processes. Organometallic approaches to cysteine S-arylation have been explored that feature many advantages compared to their more traditional organic counterparts. In this Viewpoint, progress in the use of Au(III) and Pd(II) oxidative addition (OA) complexes for stoichiometric cysteine S-arylation is presented and discussed. A focus is placed on understanding the rapid kinetics of these reactions under mild conditions, as well as the ability to generate biomolecular heterostructures. Potential avenues for further exploration are addressed and usefulness of these methods to the practitioner are emphasized in the discussion.

Investigating cholesterol trafficking pathways continues to be of significant scientific interest owing to its homeostasis being associated with several debilitating cardiovascular and neurodegenerative diseases including atherosclerosis, Niemann-Pick's disease, Alzheimer's disease, and Parkinson's disease. To further our understanding of cholesterol trafficking, it is imperative to develop new fluorescent probes that possess improved photostability, low efflux, and high spatial and temporal resolution for live-cell imaging. In this study, we developed a photoconvertible fluorescent cholesterol analog, Duo-Chol, enabling the improved spatiotemporal fluorescence imaging of the dynamic localization of cholesterol in live cells. This tool provides a unique and powerful approach to interrogating cholesterol dynamics, addressing the limitations of existing methods, and expanding our ability to probe the biological role of sterols in living cells.