Bioconjugate Chemistry最新文献

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 (CAD^C6F13F) for the efficient and safe delivery of mRNAs into cells. The PFA-modified artificial viral capsid was formed by the self-assembly of the CAD^C6F13F-conjugated β-annulus peptide, unmodified β-annulus, and dT₂₀-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.

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 ⁶⁸Ga-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 LP1 and LP2 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 EC₅₀ values: ^natGa-LP1-oxytocin (376 nM), ^natGa-DOTA-Lys⁸-oxytocin (1.38 nM), and ^natGa-LP2-oxytocin (123 nM). Radiolabeling with ⁶⁸Ga 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 ⁶⁸Ga-LP1-oxytocin (SUV_max 0.64 ± 0.10; n = 3) and ⁶⁸Ga-LP2-oxytocin (SUV_max 0.64 ± 0.05; n = 7) at 10 min postinjection, whereas ⁶⁸Ga-DOTA-Lys⁸-oxytocin reached comparable uptake (SUV_max 0.64 ± 0.12; n = 3) at 30 min. Notably, ⁶⁸Ga-LP2-oxytocin 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 ⁶⁸Ga-LP2-oxytocin 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, ⁶⁸Ga-LP2-oxytocin holds promise as a PET radioligand for targeting OTR in breast cancer.

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.

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.

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 C_H2 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.

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-R₅ 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.

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.

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 Salmonella 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.

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 (IC₅₀) 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.