Bioconjugate Chemistry最新文献

Transferrin receptor 1 (TfR1) is a ubiquitously expressed receptor characterized by rapid internalization kinetics and efficient receptor recycling, making it an attractive target for drug delivery. Herein, we investigated the potential of TfR1-binding peptide-siRNA conjugates for central nervous system (CNS)-specific gene silencing. A panel of TfR1-binding peptides and conjugation linkers were synthesized to enable siRNA attachment and evaluate their gene-silencing effects. Conjugation with the hTfR No. 894 peptide achieved effective siRNA delivery both in vitro and in vivo. Compared to ribose 2'-O-hexadecyl (C16)-siRNA conjugates, the hTfR No. 894-siRNA conjugation (POC2) elicited favorable pharmacokinetic characteristics and robust and durable silencing of the target gene across CNS regions following local administration, with minimal impact on peripheral tissues. These findings support TfR1-binding peptide conjugation as a promising strategy for CNS-targeted siRNA delivery.

Mirror-image proteins (d-proteins) are promising therapeutic molecules with high biological stability and low immunogenicity. We recently developed a novel d-monobody scaffold variant with reduced immunogenicity. This variant incorporates two cysteine substitutions that enable the chemical synthesis of d-monobodies via native chemical ligation. In this study, the structure-activity relationship of monobody scaffold variants was investigated to identify more suitable positions for cysteine modifications. Several monobody variants with different cysteine substitution patterns and additional cysteine-selective modifications were designed and synthesized. Comprehensive functional analysis of the synthetic monobody derivatives led to the identification of a favorable monobody scaffold with potent target binding and high thermal stability. The optimized monobody scaffold with a cysteine cross-linker was used to develop d-monobody with additional functional groups.

Pancreatic cancer remains one of the most aggressive malignancies with a poor prognosis due to late-stage diagnosis and limited treatment options. Fluorescence imaging has emerged as a valuable tool for early detection and targeted imaging of pancreatic cancer, offering improved visualization of tumors at the molecular level. Among various fluorescence techniques, fluorescence imaging using longer-wavelength nanomaterials holds significant promise due to their deeper tissue penetration and reduced background autofluorescence. In this study, we report the development of red-emitting carbon quantum dots designed for targeted imaging of pancreatic cancer. These carbon quantum dots were functionalized with erlotinib to enhance cancer cell specificity. In vitro biological evaluations demonstrated minimal cytotoxicity, prompting further investigations in vivo. Using BALB/c mice as model organisms, in vivo imaging showcased the efficacy of the developed probe for targeted pancreatic cancer detection, suggesting its potential as a robust tool for cancer diagnostics and imaging.

The complement system is essential for immune defense, but its dysregulation contributes to various complement-mediated disorders, including paroxysmal nocturnal hemoglobinuria (PNH). CP40 (a cyclic peptide also known as AMY101), effectively inhibits complement activation by preventing the initial binding of the C3 substrate to convertase. Despite its potency, CP40 has a very short plasma half-life when unbound to human C3, necessitating frequent dosing. We developed a novel PASylation-Lipidation Modular (PLM) platform. This platform incorporates a solubilizing PAS module and a half-life-extending lipid moiety into CP40 via a chemical linker. Systematic optimization of the spacer and lipid components in PLM-modified CP40 analogues identified 6C1 as a lead compound. Compared to CP40, 6C1 exhibited a 5-fold increase in antihemolytic potency in the classical complement pathway and a 6.3-fold improvement in solubility. In vivo studies demonstrated that PLM-CP40 analogues possess superior pharmacokinetic properties, with a 15.6-fold extension in half-life relative to unmodified CP40. Mechanistic studies revealed that the PLM platform extends half-life by interacting with albumin, which serves as a circulating depot for the compound. Surface plasmon resonance analysis and hemolysis assays postalbumin incubation demonstrated that PLM modifications maintain receptor affinity by strategically positioning the albumin-binding moiety away from the peptide region, preserving its biological activity. In a clinically relevant in vitro model of complement-mediated hemolysis in PNH, 6C1 effectively reduced erythrocyte lysis. The PLM platform thus offers a versatile strategy for enhancing peptide therapeutics by improving solubility, extending circulation time, and increasing efficacy, broadening their therapeutic potential.

Poloxamers are widely used in biomedical applications, but their effectiveness depends on achieving an optimal sol-gel phase transition near body temperature. This study evaluates three different poloxamer mixtures for their potential in treating meniscus tears, focusing on gel formation, injectability, and cell compatibility. The rheological properties, cytotoxicity assessments, and cellular migration experiments were studied using NIH/3T3 fibroblast cells as the standard experimental model for primary research. The poloxamer hydrogels showed properties well suited to injectable or drug delivery systems. Specifically, the combination of Synperonic F108 and Poloxamer 188 tended to show less adhesion and more aggregation, followed by a greater number of viable cells, suggesting its utility as a coating or foundational matrix. Concurrently, the Kolliphor 407 and Poloxamer 188 combination exhibited increased viscosity, maintaining a gel state at physiological temperature. Its biocompatibility indicated the potential for injectable controlled-release systems for musculoskeletal injuries. Our findings demonstrate that the poloxamer concentration and composition significantly influence their biomedical applications. These triblock copolymer systems indicated useful characteristics for surgical applications, such as favorable sol-gel transition kinetics and biocompatibility, suggesting potential applications in osteoarticular regeneration.

The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction can be used to modify the surface of bacteria for a variety of applications including drug delivery, biosensing, and imaging. This is usually accomplished by first installing a small azide group within the peptidoglycan and then delivering exogenous cargo (e.g., a protein or nanoparticle) modified with a cyclooctyne group, such as dibenzocyclooctyne (DBCO), for in situ conjugation. However, DBCO is comparatively bulky and hydrophobic, increasing the propensity of some payloads to aggregate. In this study, we sought to invert this paradigm by exploring two novel strategies for incorporating DBCO into the peptidoglycan of Staphylococcus aureus and compared them to an established approach using DBCO-vancomycin. We demonstrate that DBCO-modified small molecules belonging to all three classes─a sortase peptide substrate (LPETG), two d-alanine derivatives, and vancomycin─can selectively label the S. aureus surface to varying degrees. In contrast to DBCO-vancomycin, the DBCO-d-alanine variants do not adversely affect the growth of S. aureus or lead to off-target labeling or toxicity in HEK293T or RAW 264.7 cells. Finally, we show that, unlike IgG3-Fc labeled with DBCO groups, IgG3-Fc labeled with azide groups is stable (i.e., remains water-soluble) under normal storage conditions, retains its ability to bind the immune receptor CD64, and can be successfully attached to the surface of DBCO-modified S. aureus. We believe that the labeling strategies explored herein will expand the paradigm of specific, nontoxic SPAAC-mediated labeling of the surface of S. aureus and other Gram-positive bacteria, opening the door for new applications using azide-modified cargo.

Supramolecular assemblies hold great potential as biomaterials for several biomedical applications. The modification of supramolecular biomaterials is needed to achieve controlled bioactive functions. Supramolecular ureidopyrimidinone (UPy) monomers have been shown to assemble into long supramolecular polymers that can be functionalized with bioactive peptides and visualized as UPy-fibers. So far, the introduction of biological functionality has been limited to small molecules and peptides. Here, we describe a general method based on SpyTag-SpyCatcher chemistry for conjugating full-length proteins with biologically relevant functions to μm-long UPy fibers via native peptide bond formation, yielding 100% conversion in a 5:95 mol % coassembly of UPy-SpyTag with UPy-glycinamide. The conjugation of monoclonal antibodies is performed using photo-cross-linkable protein G domains. We demonstrate intact fibers and colocalization of antibodies and UPy-fibers using biophysical and imaging methods and achieve recruitment of supramolecular assemblies to the surface of mammalian cells via the EGFR-specific antibody Cetuximab. The approach introduced here represents a robust and widely applicable postassembly modification method that shows promise in the functionalization of future biomaterials.

Targeting molecules, such as antibodies and peptides, play a key role in the precise delivery of cytotoxic payloads to tumor sites by binding to specific tumor-associated antigens or other proteins within the tumor microenvironment. This investigation evaluates the potential therapeutic application of a bispecific antibody (BsAb), which simultaneously targets EphA2, a tumor-associated antigen, and CD11b, a protein expressed by tumor-associated macrophages and myeloid-derived suppressor cells (TAMCs). Recombinantly produced anti-EphA2-CD11b-BsAb was conjugated to a bifunctional chelator, NOTA-SCN, and then radiolabeled with copper-64 (⁶⁴Cu). The [⁶⁴Cu]Cu-NOTA-anti-EphA2-CD11b-BsAb radioimmunoconjugate was subsequently administered to HT1080-fibrosarcoma-bearing nude mice via tail vein injection. Positron Emission Tomography (PET) and ex vivo biodistribution analyses were performed to determine tumor uptake and pharmacokinetic localization. At 4, 24, and 48 h postinjection (p.i.), the percent injected dose per gram (%ID/g) of [⁶⁴Cu]Cu-NOTA-anti-EphA2-CD11b-BsAb in HT1080 xenografts were 5.35 ± 2.24, 4.44 ± 1.90, and 4.10 ± 0.60, respectively. There was high uptake in the liver as well as in CD11b-expressing organs, including the spleen, bone marrow, and lung. Binding in these CD11b-rich organs was significantly reduced by coadministering the dose with nonradiolabeled anti-CD11b-IgG and anti-EphA2-CD11b-BsAb, with a concurrent increase in tumor uptake compared to nonblocked mice (8.39 ± 1.37%ID/g for blocked and 4.44 ± 1.90%ID/g for nonblocked at 24 h p.i., p = 0.0175). Further optimization studies showed that at lower molar activity (3.7 MBq/nmol, 100 μCi/nmol), there were significantly higher tumor accumulations and reduced uptake in CD11b-expressing organs compared to higher molar activity (22.2 MBq/nmol, 600 μCi/nmol). Anti-EphA2-CD11b-BsAb is a functional targeting molecule and would require optimization through molar activity or blocking with nonradiolabeled antibody to maximize tumor targeting.

Oligonucleotide therapeutics (ONT) traditionally involve a single targeting moiety per oligonucleotide when conjugated for organ delivery. Multimerization represents a novel approach by connecting multiple ONTs to a single scaffold, thereby influencing the drug's activity and biophysical properties in vivo. Recently, others have demonstrated the efficacy of this strategy, showing enhanced tissue retention and extended silencing with the capability to target multiple genes simultaneously. The investigation of diverse multimeric designs is thus an exciting opportunity to explore the delivery of the ONT. In this study, we engineered a versatile peptide branching unit able to link up to four small interfering RNAs together. We conjugated a GalNAc targeting moiety to these scaffolds for liver hepatocyte delivery and assessed their silencing activity. Our approach was further expanded to explore different peptide architectures (linear versus cyclized) and additional functionalities, including endosomal escape domains and dual target silencing. We then evaluated the constructs via subcutaneous and intravenous (i.v.) administration in mice. Notably, the intravenous administration of multimeric siRNA GalNAc demonstrated potent silencing in the liver and significantly affected liver-to-kidney biodistribution. Our findings suggest that peptides as branching units offer a promising pathway for ONT multimerization, advancing the challenges of drug delivery.

Targeted delivery of cytostatic drugs is a powerful approach to achieving tumor tissue selectivity, reducing systemic toxicity, and ultimately improving the efficacy of anticancer chemotherapy. Targeting can be achieved using a wide range of molecular ligands, with DNA aptamers being a promising representative. In this work, we employed flow cytometry, a AuNP-aptasensor, and atomic-scale computer modeling to assess the affinity of several DNA aptamers (Anti-HER2, HB5, Apt-6, HeA2_1, and HeA2_3) for human epidermal growth factor receptor 2 (HER2), which is known to be one of the factors that promote the growth of breast cancer cells. Flow cytometry showed that short aptamers (HeA2_1 and HeA2_3) had a higher affinity for HER2 on MDAMB453 cancer cells than longer aptamers (HB5, Apt-6). HER2-negative MDA-MB-231 cells served as the negative control. The HeA2_3 aptamer has a high average affinity (HeA2_3:23.6, HeA2_1:13.1, Apt-6:3.6; HB5:3.5; Anti-HER2:3.2) and a nearly Gaussian distribution across the cells, while HeA2_1 forms a fraction of cells with a relatively high fluorescence signal intensity (HeA2_1:11.6; HeA2_3:5.9; Apt-6:3.4; HB5:3.1; Anti-HER2:2.1). Most of the findings for cancer cells also hold for the HER2-positive small extracellular vesicles studied using the AuNP-aptasensor. Computer simulations confirmed that short aptamers are characterized by stronger binding to the extracellular domain of HER2. A detailed analysis of the free energy allowed us to show for the first time that tight binding to HER2 correlates with well-separated hot and cold spots on the protein surface. For the aptamers that meet these criteria (HeA2_1, HeA2_3, and Anti-HER2), favorable interactions with HER2 are driven by the local attraction of nucleotides to arginine and lysine residues of HER2 and possibly stabilized by intermolecular hydrogen bonds. For longer aptamers (Apt-6 and HB5), hot and cold spots on the HER2 surface overlap and the aptamers show much weaker binding. Overall, our findings show that binding of DNA aptamers to HER2 cannot be characterized merely by the dissociation equilibrium constant. A more sophisticated approach that combines experimental and computational methods allowed us to unlock the molecular mechanisms behind the aptamer-HER2 bindings. The results of our study also suggest that computer modeling has become a reliable and accurate tool for aptamer prescreening prior to laboratory experiments.