Bioconjugate Chemistry Bioconjugate最新文献

The SpyCatcher/SpyTag system is a protein pair that forms a covalent isopeptide bond without an additional energy supply. The ability to connect fused proteins makes this system an attractive tool for several protein engineering applications. Conditional activation of the SpyCatcher/SpyTag complex formation further expands the use of this system. Here, we evaluated the pH activation of SpyTag using alkoxyaspartic acids in the isopeptide-forming residue. We found that a peptide with an ethoxy group can be activated by hydrolysis under high pH conditions. However, the hydrolysis induces isoaspartate (isoAsp) formation, which is confirmed by an isoAsp-inserted short peptide. We overcame this problem by changing the C-terminal side of the aspartic acid position to Pro, which does not form isoAsp under high pH conditions. The findings of this study provide fundamental knowledge of the synthetic construction of the modified SpyTag peptide.

Nanodiamonds (NDs) are considered promising delivery platforms, but inaccurate and uncontrolled release of drugs at target sites is the biggest challenge of NDs in precision medicine. This study presents the development of phototriggerable ND-based drug delivery systems, utilizing ortho-nitrobenzyl (o-NB) molecules as photocleavable linkers between drugs and nanocarriers. UV irradiation specifically cleaved o-NB molecules and then was followed by releasing antisense oligonucleotides from ND-based carriers in both buffer and cellular environments. This ND system carried cell nonpermeable therapeutic agents for bypassing lysosomal trapping and degradation. The presence of fluorescent nitrogen-vacancy centers also allowed NDs to serve as biological probes for tracing in cells. We successfully demonstrated phototriggered release of antisense oligonucleotides from ND-based nanocarriers, reactivating their antisense functions. This highlights the potential of NDs, photocleavable linkers, and light stimuli to create advanced drug delivery systems for controlled drug release in disease therapy, opening possibilities for targeted and personalized treatments.

Zirconium-89 is the most widely used radioisotope for immunoPET because its physical half-life (78.2 h) suits the one of antibodies. Desferrioxamine B (DFO) is the standard chelator for the complexation of zirconium(IV), and its bifunctional version, containing a phenylisothiocyanate function, is the most commonly used for the conjugation of DFO to proteins. However, preliminary results have shown that the thiourea link obtained from the conjugation of isothiocyanate and lysines is sensitive to the ionizing radiation generated by the radioisotope, leading to the rupture of the link and the release of the chelator/radiometal complex. This radiolysis phenomenon could produce nonspecific signal and prevent the detection of bone metastasis, as free zirconium accumulates into the bones. The aim of this work was to study the stability of a selection of conjugation linkers in ⁸⁹Zr-labeled immunoconjugates. We have synthesized several DFO-based bifunctional chelators appended with an isothiocyanate moiety, a bicyclononyne, or a squaramate ester. Two antibodies (trastuzumab and rituximab) were conjugated and radiolabeled with zirconium-89. The effect of increasing activities of zirconium-89 on the integrity of the bioconjugate bearing thiourea links was evaluated as well as the impact of the presence of a radioprotectant. The stability of the radiolabeled antibodies was studied over 7 days in PBS and human plasma. Radioconjugates’ integrity was evaluated using iTLC and size-exclusion chromatography. This study shows that the nature of the linker between the chelator and biomolecule can have a strong impact on the stability of the ⁸⁹Zr-labeled conjugates, as well as on the aggregation of the conjugates.

The delivery of proteins into the cytosol holds great promise for cell signaling manipulation and the development of precision medicine. However, this potency is challenged by achieving targeted and controlled delivery, specifically within diseased cells. In this study, we introduce a versatile and effective method for the precision delivery of therapeutic proteins to cancer cells by designing polyphenol-assisted biomineralization of zeolite imidazole framework-8 (ZIF-8). We demonstrate that by leveraging the strong noncovalent binding affinity of epigallocatechin gallate (EGCG) with both proteins and ZIF-8, our approach significantly enhances the biomineralization of ZIF-8, which in turn improves the efficiency of protein encapsulation and intracellular delivery. Moreover, the incorporation of EGCG within ZIF-8 enables controlled degradation of the nanoparticles and the selective release of the encapsulated proteins in cancer cells. This selective release is triggered by the oxidation of EGCG in response to the high levels of reactive oxygen species (ROS) found within cancer cells that destabilize the EGCG/ZIF-8 nanoparticles. We have further demonstrated the ability of EGCG/ZIF-8 to deliver a wide range of proteins into cancer cells, including bacterial virulence protein, to rewire cell signaling and prohibit tumor cell growth in a mouse xenograft model. Our strategy and findings underscore the potential of designing the EGCG/ZIF-8 interface for specific and controlled protein delivery for targeted cancer therapy.

The exploration of pharmaceutically active agents and positron emission tomography (PET) tracers targeting CXCR4 has been a focal point in cancer research given its pivotal role in the development and progression of various cancers. While significant strides have been made in PET imaging with radiometal-labeled tracers, the landscape of ¹⁸F-labeled small molecule tracers remains relatively limited. Herein, we introduce a novel and promising derivative, [¹⁸F]SFB-AMD3465, as a targeted PET tracer for CXCR4. The compound was synthesized by modifying the pyridine ring of AMD3465, which was subsequently labeled with ¹⁸F using [¹⁸F]SFB. The study provides comprehensive insights into the design, synthesis, and biological evaluation of [¹⁸F]SFB-AMD3465. In vitro and in vivo assessments demonstrated the CXCR4-dependent, specific, and sensitive uptake of [¹⁸F]SFB-AMD3465 in the CXCR4-overexpressing 4T1 cell line and the corresponding xenograft-bearing mouse model. These findings contribute to bridging the gap in ¹⁸F-labeled PET tracers for CXCR4 and underscore the potential of [¹⁸F]SFB-AMD3465 as a PET radiotracer for in vivo CXCR4 imaging.

Addressing the complex challenge of healing of bacterially infected wounds, this study explores the potential of lipid nanomaterials, particularly advanced ultradeformable particles (UDPs), to actively influence the wound microenvironment. The research introduces a novel therapeutic approach utilizing silver sulfadiazine (SSD) coupled with vitamin E (VE) delivered through UDPs (ethosomes/transferosomes/transethosomes). Comparative physicochemical characterization of these nanosized drug carriers reveals the superior stability of transethosomes, boasting a zeta potential of −36.5 mV. This method demonstrates reduced side effects compared to conventional therapies, with almost 90% SSD and 72% VE release achieved in wound pH in a sustained manner. Cytotoxicity assessment shows 60% cell viability even at the highest concentration (175 μg/mL), while hemolysis test demonstrates RBC lysis below 5% at a concentration of 250 μg/mL. Vitamin E–SSD-loaded transethosomes (VSTEs) significantly enhance cellular migration and proliferation, achieving 95% closure within 24 h, underscoring their promising efficacy. The synergistic method effectively reduces bacterial burden, evidenced by an 80% reduction in Escherichia coli and Staphylococcus aureus within the wound microenvironment. This approach offers a promising strategy to address complications associated with skin injuries.

Enhancing the accumulation and retention of small-molecule probes in tumors is an important way to achieve accurate cancer diagnosis and therapy. Enzyme-stimulated macrocyclization of small molecules possesses great potential for enhanced positron emission tomography (PET) imaging of tumors. Herein, we reported an ¹⁸F-labeled radiotracer [¹⁸F]AlF-RSM for legumain detection in vivo. The tracer was prepared by a one-step aluminum-fluoride-restrained complexing agent ([¹⁸F]AlF-RESCA) method with high radiochemical yield (RCY) (88.35 ± 3.93%) and radiochemical purity (RCP) (>95%). More notably, the tracer can be transformed into a hydrophobic macrocyclic molecule under the joint action of legumain and reductant. Simultaneously, the tracer could target legumain-positive tumors and enhance accumulation and retention in tumors, resulting in the amplification of PET imaging signals. The enhancement of radioactivity enables PET imaging of legumain activity with high specificity. We envision that, by combining this highly efficient ¹⁸F-labeled strategy with our intramolecular macrocyclization reaction, a range of radiofluorinated tracers can be designed for tumor PET imaging and early cancer diagnosis in the future.

Disorder of complement response is a significant pathogenic factor causing some autoimmune and inflammation diseases. The Ornithodoros moubata Complement Inhibitor (OmCI), a small 17 kDa natural protein, was initially extracted from soft tick salivary glands. The protein was found binding to complement C5 specifically, inhibiting the activation of the complement pathway, which is a successful therapeutic basis of complement-mediated diseases. However, a short half-life due to rapid renal clearance is a common limitation of small proteins for clinical application. In this study, we extended the half-life of OmCI by modifying it with fatty acid, which was a method used to improve the pharmacokinetics of native peptides and proteins. Five OmCI mutants were initially designed, and single-site cysteine mutation was introduced to each of them. After purification, four OmCI mutants were obtained that showed similar in vitro biological activities. Three mutants of them were subsequently coupled with different fatty acids by nucleophilic substitution. In total, 15 modified derivatives were screened and tested for anticomplement activity in vitro. The results showed that coupling with fatty acid would not significantly affect their complement-inhibitory activity (CH50 and AH50). OmCIT90C-CM02 and OmCIT90C-CM05 were validated as the applicable OmCI bioconjugates for further pharmacokinetic assessments, and both showed improved plasma half-life in mice compared with unmodified OmCI (15.86, 17.96 vs 2.57 h). In summary, our data demonstrated that OmCI conjugated with fatty acid could be developed as the potential long-acting C5 complement inhibitor in the clinic.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have been approved for once or twice daily oral use in the treatment of cancers with BRCA defects. However, for some patients, oral administration of PARPi may be impractical or intolerable, and a long-acting injectable formulation is desirable. We recently developed a long-acting PEGylated PARPi prodrug, PEG∼talazoparib (TLZ), which suppressed the growth of PARPi-sensitive tumors in mice for very long periods. However, the release rate of TLZ from the conjugate was too fast to be optimal in humans. We prepared several new PEG∼TLZ prodrugs having longer half-lives of drug release and accurately measured their pharmacokinetics in the rat. Using the rates of release of TLZ from these prodrugs and the known pharmacokinetics of free TLZ in humans, we simulated the pharmacokinetics of the macromolecular prodrugs and released TLZ in humans. From several possibilities, we chose two conjugates that could be administered intravenously every 2 weeks and maintain TLZ within its known therapeutic window. We describe situations where the PEG∼TLZ conjugates would find utility in humans and suggest how the intravenously administered long-acting prodrugs could in fact be more effective than daily oral administration of free TLZ.