Immobilization of glucose oxidase on bioinspired polyphenol coatings as a high-throughput glucose assay platform.

Glucose oxidase (GOx) is an enzyme with important industrial and biochemical applications, particularly in glucose detection. Here we leveraged the oxidative self-polymerization phenomenon of simple polyphenols (pyrogallol or catechol) in the presence of polyethylenimine (PEI) to form adhesive coatings that enabled GOx immobilization on conventional multi-well plates. Immobilization was verified and optimized by directly measuring GOx activity inside the coated wells. Our results showed that incorporating PEI in polyphenol coatings enhanced their enzyme immobilization efficiency, with pyrogallol (PG)-based coatings displaying the greatest enzyme activity. The immobilized enzyme maintained similar affinity to glucose compared to the free enzyme. GOx-immobilized PG/PEI-coated wells exhibited intermediate recycling ability but excellent resistance to urea as a denaturing agent compared to the free enzyme. GOx-immobilized 96-well plates allowed the construction of a linear glucose calibration curve upon adding glucose standards, with a detection limit of 0.4-112.6 mg dL-1, which was comparable to commercially available enzymatic glucose assay kits. The assay platform was also capable of effectively detecting glucose in rat plasma samples. Our findings present a simple enzyme immobilization technique that can be used to construct a glucose assay platform in a convenient multi-well format for high-throughput glucose quantification.

Development of a Thymoquinone Polymeric Anticancer Nanomedicine through Optimization of Polymer Molecular Weight and Nanoparticle Architecture.

Thymoquinone (TQ) is a water-insoluble natural compound isolated from Nigella sativa that has demonstrated promising chemotherapeutic activity. The purpose of this study was to develop a polymeric nanoscale formulation for TQ to circumvent its delivery challenges. TQ-encapsulated nanoparticles (NPs) were fabricated using methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) (mPEG-PCL) copolymers by the nanoprecipitation technique. Formulation variables included PCL chain length and NP architecture (matrix-type nanospheres or reservoir-type nanocapsules). The formulations were characterized in terms of their particle size, polydispersity index (PDI), drug loading efficiency, and drug release. An optimized TQ NP formulation in the form of oil-filled nanocapsules (F2-NC) was obtained with a mean hydrodynamic diameter of 117 nm, PDI of 0.16, about 60% loading efficiency, and sustained in vitro drug release. The formulation was then tested in cultured human cancer cell lines to verify its antiproliferative efficacy as a potential anticancer nanomedicine. A pilot pharmacokinetic study was also carried out in healthy mice to evaluate the oral bioavailability of the optimized formulation, which revealed a significant increase in the maximum plasma concentration (Cmax) and a 1.3-fold increase in bioavailability compared to free TQ. Our findings demonstrate that the versatility of polymeric NPs can be effectively applied to design a nanoscale delivery platform for TQ that can overcome its biopharmaceutical limitations.

Nature-Inspired Polymerization of Quercetin to Produce Antioxidant Nanoparticles with Controlled Size and Skin Tone-Matching Colors.

Plant polyphenols have received considerable attention in recent years due to their ability to undergo oxidation-triggered self-polymerization, forming biocompatible versatile coatings and templated nanoparticles (NPs) that can be leveraged for a variety of biomedical applications. Here we show for the first time that untemplated NPs can be conveniently synthesized from the abundant plant polyphenol quercetin (QCT) simply by incubation with an oxidizing agent in a universal organic solvent, followed by self-assembly upon gradual addition of water. The process yielded NPs of around 180-200 nm in size with a range of colors that resembled light to medium-brown skin tones. The NPs were characterized by UV-Vis, FT-IR, and 1H-NMR spectroscopy and by dynamic light scattering and transmission electron microscopy to understand their physicochemical properties. Antioxidant and cell viability assays were also conducted to demonstrate the NPs' free-radical scavenging activity and biocompatibility, altogether providing valuable insights into the structure and function of this emerging class of nanomaterials to guide future biomedical applications.

Bioinspired Polymerization of Quercetin to Produce a Curcumin-Loaded Nanomedicine with Potent Cytotoxicity and Cancer-Targeting Potential in Vivo.

Nanomedicine has had a profound impact on the treatment of many diseases, especially cancer. However, synthesis of multifunctional nanoscale drug carriers often requires multistep coupling and purification reactions, which can pose major scale-up challenges. Here, we leveraged bioinspired oxidation-triggered polymerization of catechols to synthesize nanoparticles (NPs) from the plant polyphenol quercetin (QCT) loaded with a hydrophobic anticancer drug, curcumin, and functionalized with poly(ethylene glycol) (PEG) for steric stabilization in one reaction step. NPs were formed by base-catalyzed oxidative self-polymerization of QCT in the presence of curcumin and thiol-terminated PEG upon mixing in a universal solvent (dimethyl sulfoxide), followed by self-assembly with the gradual addition of water. Dynamic light scattering and X-ray photoelectron spectroscopy were used to confirm NP PEGylation. Drug loading was verified by UV-vis spectroscopy. Curcumin-loaded NPs were efficiently internalized by CT26 murine colon cancer cells as determined by flow cytometry and confocal microscopy. NPs also demonstrated sustained release and potent cytotoxicity in vitro. Moreover, in vivo imaging of CT26 tumor-bearing Balb/c mice following tail vein injection of DiR-labeled QCT NPs showed steady tumor accumulation of the NPs up to 24 h. This was further supported by significant tumor uptake of curcumin-loaded QCT NPs as measured by flow cytometry analysis of tumor homogenates. Our findings present a greener synthetic route for the fabrication of drug-loaded surface-functionalized NPs from poorly water-soluble plant polyphenols such as QCT as promising anticancer delivery systems.