Selective Conjugation of Proteins by Mining Active Proteomes through Click-Functionalized Magnetic Nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) coated with azide groups were functionalized at the surface with biotin (biotin@SPIONs) and cysteine protease inhibitor E-64 (E-64@SPION5) with the purpose of developing nanoparticle-based assays for identifying cysteine proteases in proteomes. Magnetite particles (ca. 6 nm) were synthesized by microwave-assisted thermal decomposition of iron acetylacetonate and subsequently functionalized following a click chemistry protocol to obtain biotin and E-64 labeled particulate systems. Successful surface modification and covalent attachment of functional groups and molecules were confirmed by FT-IR spectroscopy and thermal gravimetric analysis. The ability of the surface-grafted biotin terminal groups to specifically interact with streptavidin (either horseradish peroxidase [(HRP)-luminol-H2O2] or rhodamine) was confirmed by chemiluminescent assay. A quantitative assessment showed a capture limit of 0.55-1.65 mu g protein/100 mu g particles. Furthermore, E-64@SPION5 were successfully used to specifically label papain-like cysteine proteases from crude plant extracts. Owing to the simplicity and versatility of the technique, together with the superparamagnetic behavior of FeOx-nanoparticles, the results demonstrate that click chemistry on surface anchored azide group is a viable approach toward bioconjugations that can be extended to other nanoparticles surfaces with different functional groups to target specific therapeutic and diagnostic applications

Reversible Covalent Assembly of Nanoparticles through On-Surface Diets-Alder Reaction

We demonstrate here a controlled assembly of individual nanoscale building blocks into defined architectures based on chemospecific covalent bonding interactions. For this purpose, alpha-Fe2O3, gamma-Fe2O3, and SiO2 nanoparticles decorated with surface-conjugated organic ligands were used for performing on-surface Diets-Alder reactions. Driven through their chemical affinity and surface-grafted complementary functionalities, nanoparticles underwent click-reactions to produce covalently organized nanostructures. An advantage of using the Diels-Alder reaction is its reversible nature, which was used to click and unclick the nanoparticles on demand. The efficiency and chemical specificity of this approach opens up another synthetic access to unify materials with complementary properties, where the thermoresponsive nature of particle assemblies imparts to them a fully reversible character. The covalent conjugation strategies demonstrated in this work potentially allow the use of a diverse range of particles and ligands for their applications in different disciplines such as medicine, optics, or photonics. The nanoparticles morphology and crystalline nature were investigated by TEM and XRD analysis, while the presence of surface attached groups was verified by NMR, FTIR, UV-vis, and potential measurements

Multivalent magnetic nanoaggregates with unified antibacterial activity and selective uptake of heavy metals and organic pollutants

Covalently functionalized magnetite (Fe3O4) nanoaggregates carrying an imidazolium-derivative (1-hexadecyl-3-vinyl imidazolium bromide, HDVI) and L-cysteine (L-Cys) as surface ligands act as bimodal water-treatment particulate agents (HDVI@L-Cys@PAA@Fe3O4) with high antibacterial efficacy and specific surface adsorption properties. For covalent conjugation of HDVI the polyacrylic acid (PAA)-coated magnetite nanocrystals (PAA@Fe3O4) were functionalized with L-cysteine via carbodiimide coupling (L-Cys@PAA@Fe3O4) having terminal-SH groups that were used for thiol-ene click chemistry. The carefully performed series of water-remediation tests with magnetically separable HDVI@L-Cys@PAA@Fe3O4 nanoaggregates demonstrated their high efficiency in the concomitant removal transition metal ions and organic pollutant without losing the antibacterial effect. Time-dependent adsorption experiments showed high degree (>90%) of trapping and removal activity. Antibacterial action of the HDVI@L-Cys@PAA@Fe3O4 nanoaggregates originates from the amphiphilic structure of HDVI groups capable of penetrating the bacterial cell walls. The presence of surface-bound ligands and conversion efficiency of carbodiimide and click chemistry protocol was verified by FT-IR, elemental and thermogravimetric analysis. The phase, composition morphology and surface charge of nanoaggregates were examined by XRD, SEM/TEM and zeta potential studies, respectively. The experimental findings reported here represent a conceptual advancement in the state-of-the-art magnetic beads developed for water purification or remediation purposes. Our results evidently demonstrate that nanoaggregates are highly effective in unifying bactericidal activity against different microorganism with heavy metal and organic pollutant removal properties. (C) 2020 Elsevier B.V. All rights reserved

Selective Conjugation of Proteins by Mining Active Proteomes through Click-Functionalized Magnetic Nanoparticles

<u>S</u>uper<u>p</u>aramagnetic iron <u>o</u>xide <u>n</u>anoparticles (SPIONs) coated with azide groups were functionalized at the surface with biotin (biotin@SPIONs) and cysteine protease inhibitor E-64 (E-64@SPIONs) with the purpose of developing nanoparticle-based assays for identifying cysteine proteases in proteomes. Magnetite particles (<i>ca.</i> 6 nm) were synthesized by microwave-assisted thermal decomposition of iron acetylacetonate and subsequently functionalized following a click chemistry protocol to obtain biotin and E-64 labeled particulate systems. Successful surface modification and covalent attachment of functional groups and molecules were confirmed by FT-IR spectroscopy and thermal gravimetric analysis. The ability of the surface-grafted biotin terminal groups to specifically interact with streptavidin (either horseradish peroxidase [(HRP)-luminol-H₂O₂] or rhodamine) was confirmed by chemiluminescent assay. A quantitative assessment showed a capture limit of 0.55–1.65 μg protein/100 μg particles. Furthermore, E-64@SPIONs were successfully used to specifically label papain-like cysteine proteases from crude plant extracts. Owing to the simplicity and versatility of the technique, together with the superparamagnetic behavior of FeOx-nanoparticles, the results demonstrate that click chemistry on surface anchored azide group is a viable approach toward bioconjugations that can be extended to other nanoparticles surfaces with different functional groups to target specific therapeutic and diagnostic applications

Nanoparticle Arrays Having Directed Hybrid Topology via Covalent Self-Assembly of Iron Oxide and Silica Nanoparticles

Combining individual nanoparticles (NPs) of different chemical composition and surface chemistry offers an unexplored synthetic avenue for unifying different functionalities into a topologically defined hybrid structure. In this study, multiparticle architectures (alpha-Fe2O3@SiO2, alpha-Fe2O3@gamma-Fe2O3) were fabricated by a directed self-assembly of nanoparticles carrying complementary surface ligands suitable for click chemistry. For this purpose, the surface-rooted hydroxyl groups of freshly prepared alpha-Fe2O, gamma-Fe2O3, and SiO2 nanoparticles were utilized for facile conjugation of organic ligands, 4-(azidoacetyl)-catechol, and 10-undecynoic acid to subsequently perform copper catalyzed reactions. This work explores specific chemical interactions among functionalized nanopartides of different sizes and compositions to create nanoparticle assemblies with hybrid topologies. Here, developed on-surface chemistry protocols demonstrate that chemical specificity known for dick-chemistry protocols among molecular species is equally effective between the ligands grafted on the surface of nanoparticles. Owing to the presence of active ligands and their mutual binding affinities, architectures of nanoparticles with a high degree of symmetry and notable magnetic properties were obtained. The exterior of the nanoassembly presents active surface groups for further functionalization and attachment of modality such as the dual-mode magnetic resonance imaging. The morphology and crystalline nature of the NPs were investigated by transmission electron microscopy and X-ray diffraction analysis, whereas the active nature of the surface attached groups was determined by NMR, Fourier transform infrared, UV-vis, and zeta-potential measurements

Influence of ripening stages and drying methods on polyphenolic content and antioxidant activities of mulberry fruits

This study highlights the influence of ripening (un-ripen, semi-ripen and fully-ripen) and drying (fresh, ambient-dried and sun-dried) on the extract yield, total phenolic contents (TPC) and total flavonoid contents (TFC) of different species (Morus alba, M. nigra, M. macroura and M. laevigata) of mulberry fruit. 1,1-Diphenyl-2-picryl hydrazyl scavenging assay and reducing ability was used to assess the antioxidant capacity of the extracts. An increasing trend in the extract yield (2.9-56%), TPC (201-2287 mg/100 g GAE), TFC (58-1021 mg/100 g CE) and antioxidant activity was observed as the maturity of fruits progressed. On the other hand, fully ripened mulberry fruit at air drying conditions generally have higher antioxidant activity. The ripening stages, as well as the drying conditions has a significant impact on the polyphenols and antioxidant attributes of mulberries

Mediating the Fate of Cancer Cell Uptake: Dual-Targeted Magnetic Nanovectors with Biotin and Folate Surface Ligands

Recognition of folate and biotin surface receptors by dual-functionalized nanoparticles (NPs) is key for site-selective receptor-mediated transport of anticancer drugs to cancer cells. We present here dopamine-capped iron oxide nanoprobes (Fe3O4, 10 +/- 2 nm) containing two surface-grafted biologically relevant ligands, namely, folic acid (FA) and biotin (BT). The covalent attachment of both FA and BT on Fe3O4 nanoparticles was achieved by following carbodiimide coupling and click-chemistry protocols. The dual-function Fe 3 0 4 probes were delivered into E-G7 and human HeLa cancer cell lines and tested toward their cellular uptake by immunofluorescence and flow cytometry analysis. Owing to receptor-mediated endocytosis, enhanced accumulation of nanoprobes in cancer cells was successfully monitored by confocal laser microscopy. When compared to dual-function probes, single-functionalized nanoparticles possessing either FA or BT ligands showed significantly reduced uptake in the tested cell lines, underlining the superior interaction potential of dual-purpose probes. A time-dependent receptor-mediated endocytosis of FA-Fe3O4-BT nanovectors was demonstrated by flow cytometry analysis, whereas the unfunctionalized NPs did not show any specificity in terms of uptake. Besides their specific uptake, the surface-functionalized nanoparticles exhibited promising cytotoxicity profiles by demonstrating good viability of more than 95% with analogous cancer cell lines. Our results demonstrate that dual and/or multivariate conjugation of receptor-specific ligands on NPs is highly effective in molecular recognition of surface biomarkers that enhances their potential in anticancer treatment for pretargeting-radio strategies based on biotin/avidin interactions

Evaluation of Magnetite Nanoparticle-Based Toxicity on Embryo-Larvae Stages of Zebrafish (Danio rerio)

Iron oxide nanoparticles (NPs) are extensively used in various biomedical applications due to their suitability as carriers of diagnostic and therapeutic agents. Herein, we report on the evaluation of concentration-dependent toxicity studies of iron oxide (magnetite) NPs in zebrafish (Danio rerio). To follow the update of iron oxide NPs in the animal model, particles were functionalized by conjugating a fluorescent dye (Congo red) that serves as an efficient probe to track the uptake and accumulations of nanoparticles from the early life stages of zebrafish. As in vivo model organism to study the diffusion an in vivo toxicity, zebrafish embryos and larvae were treated with different concentrations of CR@Fe3O4 conjugates (100-800 mu g/mL) for 4-96 h postfertilization (hpf). Monitoring of mortality, hatching rate, and whole-embryo cellular death showed that incubation of low concentrations of the NPs did not exhibit adverse developmental toxicity during embryonic and larval stages of zebrafish. Minimal toxic effects were observed at high concentration (800 mu g/mL) of CR@Fe3O4, causing mortality and delay in hatching cycles. However, CR@Fe3O4 exhibited higher toxic effect on zebrafish larvae, suggesting higher bioavailability of the NPs on the tested animal stage. This study provides an investigation on developmental toxicity in zebrafish caused by Fe3O4 NPs and provides innovative insight into CR@Fe3O4 NPs serving as an optical probe to study the potential nanotoxicological effects of NPs in in vivo systems

Asymmetric attachment and functionalization of plasmonic nanoparticles on ceramic interfaces

The demands for materials that integrate more than one functional imaging or therapeutic unit are of increasing interest for biomedical applications. Here, we present the step-by-step preparation of asymmetric and optically active particles, namely, Gd2O3@Ag, Gd2O3@Au, SiO2-N-3@Au, and SiO2-SH@Au . Successful attachment of plasmonic nanoparticles to the surface of metal-oxide spheres without necessity of a potentially toxic inter-adhesive layer was proven by optical methods as well as X-ray photoelectron spectroscopy. The combination of optical and magnetic properties as present in Gd2O3@Ag and Gd2O3@Au Janus-type particles leads to dual-imaging probes for optical and magnetic resonance imaging. In addition, functional groups, such as azide groups, were linked to the surface of silica particles previous to Au nanoparticle attachment. Subsequent site-selective click reactions with 5-FAM were successfully performed as demonstrated by UV-Vis measurements. All described systems exhibited excellent long-term stability and can, therefore, be considered as promising candidates for theranostic applications. [GRAPHICS]