Bioconjugate Chemistry最新文献

This study presents the synthesis, characterization, and application of multifunctional PAMAM G2 and G4 dendrimers decorated with a linear fluorinated guanidino linker designed to improve gene delivery efficiency while minimizing cytotoxicity. For the first time, we were able to fine-tune the degree of grafting (DG) during the functionalization process through efficient "click" Michael addition, achieving the synthesis of a collection of six PAMAM conjugates that showed a significant enhancement in transfection efficiency (TE), surpassing the performance of traditional nonviral vectors. The incorporation of fluorinated moieties not only facilitated better deoxyribonucleic acid (DNA) condensation and TE but also introduced potential applications in ¹⁹F magnetic resonance imaging thanks to the sharp and intense fluorine nuclear magnetic resonance signals and favorable relaxation parameters. The new dendrimer conjugates demonstrated a promising balance between low cytotoxicity and high TE, with the low-generation PAMAM G2 with lower DG being the best-performing conjugate, making them strong candidates for further development in gene therapy. These findings highlight the potential of these multifunctional PAMAM dendrimers as efficient, nontoxic, and trackable gene delivery vectors.

To achieve the desired therapeutic response, drug delivery systems must ensure the controlled release of the loaded content at the targeted site. One possible strategy relies on the improvement of conventional drug delivery systems. To do so, smart polymers, able to change their behavior upon chemical, physical, or biological stimuli, can be used. In this context, this study aims to evaluate the potential of natural amphiphilic smart elastin-like polypeptides grafted with alkyl chains (ELP-g-Bu) to stabilize conventional oil-in-water emulsions and trigger the release of loaded molecules upon dual stimuli. With butyl pendant chains and methionine residues, the macromolecular surfactant ELP-g-Bu demonstrated a modification of physicochemical properties, looking at critical aggregation concentration, upon both temperature and oxidation stimuli. The macromolecular surfactant was then able to stabilize a paraffin-oil-in-water emulsion. The ELP-g-Bu emulsion presented a droplet size of 9 ± 1 μm and stability for at least a month at 4 and 25 °C. After successful loading of a fluorescent lipophilic molecule used as a drug model, a complete destabilization of the ELP-g-Bu emulsion and burst release of the content was achieved with thermal triggering at 42 °C. In oxidative conditions, a partial release was measured, which can be improved by increasing the number of oxidable thioether groups. Overall, these dually responsive amphiphilic ELP-g-Bu demonstrated their potential for smart-polymer-based drug delivery systems that can be promising for inflammatory disease treatment as increased temperature and radical oxygen species are present in such cases.

Fluorescent unnatural amino acids give biochemists, biophysicists, and bioengineers new ways to probe the properties of proteins and peptides. Here, the synthesis of acridon-2-ylalanine (Acd) is optimized for large-scale production to enable ribosomal incorporation through genetic code expansion (GCE), and fluorenylmethoxycarbonyl (Fmoc)-protected Acd is prepared for solid-phase peptide synthesis (SPPS). We demonstrate the utility of Acd in several applications: first, Acd quenching by Tyr is used in the design of fluorescent protease sensors made by SPPS. Second, we demonstrate Acd incorporation into a lanthanide-binding peptide that is generated either by GCE or by SPPS and demonstrate the utility of Acd for sensitizing the emission of Eu³⁺. Finally, Acd is inserted into the intrinsically disordered protein, α-synuclein, using GCE and used to study ion binding and aggregation.

The peptide–drug conjugate (PDC) has emerged as one of the new approaches for cancer therapy, which has the advantages of improved drug target ability and reduced adverse effects compared with the traditional chemotherapy. CD133 is a surface antigen specific to cancer stem cells, which are thought to be responsible for the self-renewal, proliferation, metastasis, and chemoresistance of cancer cells. A PDC for CD133 was designed by us, and it consists of CD133 targeting peptide LS-7 (amino acid sequence LQNAPRS), a pH-sensitive linker (succinyl), and a cytotoxic payload, the cytotoxic molecule camptothecin (CPT) with potent toxicity in vivo and in vitro. An antitumor study exhibited that the conjugate LS-7-CPT has not only improved its cytotoxicity in tumor cells but also retained its anticancer effect in vivo. In addition, the acute toxicity in mice of LS-7-CPT has been improved and the maximum tolerated dose has been increased by at least 56.2-fold. Pull-down and in vivo fluorescent imaging results indicated that LS-7-CPT was enriched in mice tumors by targeting CD133 protein. As far as we know, this is the first report for a PDC molecule designed for CD133, which is important for the study of CPT drug development.

Cancer vaccines are a promising strategy to increase tumor-specific immune responses in patients who do not adequately respond to checkpoint inhibitors. Cancer vaccines that contain patient-specific tumor antigens are most effective but also necessitate the production of patient-specific vaccines. This study aims to develop a versatile cancer vaccine format in which patient-specific tumor antigens can be site-specifically conjugated by a proximity-based Sortase A (SrtA)-mediated ligation (PBSL) approach to antibodies that specifically bind to antigen-presenting cells to stimulate immune responses. DEC205 and CD169 are both receptors expressed on antigen-presenting cells that can be targeted to deliver antigens and stimulate T-cell responses. We used the CRISPR/HDR platform to produce mouse heavy chain IgG2a antibodies with DEC205 or CD169 specificity containing an SrtA recognition motif followed by a SpyTag at the C-terminus. Using a recombinant protein of SrtA linked to SpyCatcher, we applied proximity-based SrtA-mediated ligation to ligate fluorescein isothiocyanate (FITC)-labeled or antigenic peptides to the antibodies. Ligated antibodies bound to DEC205-expressing dendritic cells or CD169-expressing macrophages both in vitro and in vivo. More importantly, immunization with DEC205- or CD169-specific Abs linked to T-cell epitopes efficiently stimulated T-cell responses in vivo. To conclude, we have developed a cancer vaccine format using PBSL that enables the rapid incorporation of tumor antigens and could potentially be implemented for the synthesis of personalized cancer vaccines.

Conjugates of the biocompatible polysaccharide pullulan with a cell membrane permeabilizing peptide L17E (PL-L17Es) were prepared with the aim of producing complex coacervates with pronounced intracellular antibody (IgG) delivery activity and stable structures. Coacervates with diameters of a few μm were formed simply by mixing PL-L17Es with IgG labeled with negatively charged fluorescent moieties of Alexa Fluor 488 [IgG(AF488)]. The coacervate resulted in a pronounced cytosolic infusion of IgG(AF488) and IgG binding to the target proteins inside the cell. The droplet structures were maintained even under high salt conditions, and the fluorescence in the droplet was not recovered after photobleaching, suggesting the formation of complex coacervate microgels. Dynamic changes in cell membrane structure to entrap the coacervate microgels were captured by confocal and electron microscopy, resulting in cytosolic IgG infusion. The use of M-lycotoxin instead of L17E resulted in a coacervate microgel with marked IgG delivery activity even in the presence of serum. Successful IgG delivery to primary hepatocytes, undifferentiated induced pluripotent stem (iPS) cells, and iPS cell-derived intestinal epithelial cells was also achieved. The construction of complex coacervate microgels with design flexibility and the validity of intracellular IgG delivery with high salt stability were thus demonstrated.

Fluorescent voltage indicators enable the optical recording of electrophysiology across large cell populations with subcellular resolution; however, their application is often constrained by a limited photon budget. To address this limitation, advanced bioconjugation methods have been employed to site-specifically attach bright and photostable organic dyes to cell-specific protein scaffolds in live cells. The resulting chemigenetic hybrid voltage indicators enable sustained monitoring of voltage fluctuations with an exceptional signal-to-noise ratio, both in vitro and in vivo. This Viewpoint discusses recent advancements in the development of these indicators through bioconjugation chemistry.

Immunocapture liquid chromatography–mass spectrometry (IC-LC-MS) bioanalysis has become an indispensable technique across various scientific disciplines, ranging from drug discovery to clinical diagnostics. While traditional immunocapture techniques have proven to be effective, they often encounter limitations in sensitivity, specificity, and compatibility with MS analysis. Chemoenzymatic immunocapture and protein capture (IPC) offers a promising solution, combining the high specificity of antibodies or proteins with the versatility of enzymatic and chemical modifications. This Review explores the foundational principles of chemoenzymatic IPC and examines various modification strategies including bioorthogonal click-chemistry, enzymatic-tagging, and HaloTag/CLIP-tag. Recent advancements in chemoenzymatic IPC techniques have significantly expanded their applicability to a diverse range of biomolecules including small molecules, peptides, RNAs, and proteins. This Review focuses on improvements in analytical performance achieved through these innovative approaches. Moreover, we discuss the broad applications of chemoenzymatic immunocapture in drug discovery, clinical diagnostics, and environmental analysis and explore its potential for future advancements in bioanalysis. We propose a novel solid-phase chemoenzymatic IPC assay (SCEIA) that effectively utilizes bioorthogonal click chemistry and chemoenzymatic approaches for efficient IPC and target analyte release. In summary, chemoenzymatic IPC represents a transformative paradigm shift in IC-LC-MS bioanalysis. By overcoming the limitations of traditional IPC techniques, this approach paves the way for more robust, sensitive, and versatile analytical workflows.

Because of the insidious nature of lymphatic metastatic cancer, accurate imaging tracing is very difficult to achieve in the clinic. Previous studies have developed the LARGR peptide (named TMVP1) as a radiotracer for vascular endothelial growth factor receptor-3 (VEGFR-3) imaging in cancer. However, its affinity for the target remains insufficient, resulting in low imaging sensitivity. In this study, we identified a high-affinity VEGFR-3 targeting peptide, named TMVP1446, using a multiplex screening platform. TMVP1446 demonstrated a dissociation constant of 8.97 × 10^–8 M. Both in vitro and in vivo assays confirmed that fluorescently labeled TMVP1446 specifically bound to VEGFR-3. In a 4T1-luciferase tumor mouse model, cyanine 7-labeled TMVP1446 effectively discriminated between contralateral normal lymph nodes (c-LN) and cancer-metastatic sentinel lymph nodes (m-SLN). To evaluate the potential of TMVP1446, we developed a novel VEGFR-3 positron emission tomography radiotracer ([⁶⁸Ga]Ga-DOTA-TMVP1446) for cancer-m-SLN imaging. [⁶⁸Ga]Ga-DOTA-TMVP1446 accurately detected and assessed the status of lymph node metastasis, even in micrometastatic tumors, in the B16–F10 mouse tumor model. These findings suggest that TMVP1446 has great potential for advancing VEGFR-3 molecular imaging and metastatic sentinel lymph node imaging.

Quinine is a promising building block for creating polymer carriers for intracellular nucleic acid delivery. This is due to its ability to bind to genetic material through intercalation and electrostatic interactions and the balance of hydrophobicity and hydrophilicity dependent on the pH/charge state. Yet, studies utilizing cinchona alkaloid natural products in gene delivery are limited. Herein, we present the incorporation of a quinine functionalized monomer (Q) into block polymer architectures to form self-assembled micelles for highly efficient gene delivery. Q was incorporated into the core and/or the shell of the micelles to introduce the unique advantages of quinine to the system. We found that incorporation of Q into the core of the micelle resulted in acid-induced disassembly of the micelle and a boost in transfection efficiency by promoting endosomal escape. This effect was especially evident in the cancerous cell line, A549, which has a more acidic intracellular environment. Incorporation of Q into the shell of the micelles resulted in intercalative binding to the genetic payload as well as larger micelle-DNA complexes (micelleplexes) from the hydrophobicity of Q in the shell. These factors enable the micelleplexes to be more resistant to serum and have more persistent protein expression post-transfection. Overall, this study is the first to demonstrate the benefits of including quinine functionalities into self-assembled micelles for highly efficient gene delivery and presents a platform for inclusion of other natural products with similar properties into micellar systems.