Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes.

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.

Robust Oxidase-Mimetic Supramolecular Nanocatalyst for Lignin Biodegradation.

Enzymatic catalysis presents an eco-friendly, energy-efficient method for lignin degradation. However, challenges arise due to the inherent incompatibility between enzymes and native lignin. In this work, we introduce a supramolecular catalyst composed of fluorenyl-modified amino acids and Cu2+, designed based on the aromatic stacking of the fluorenyl group, which can operate in ionic liquid environments suitable for the dissolution of native lignin. Amino acids and halide anions of ionic liquids shape the copper site's coordination sphere, showcasing remarkable catechol oxidase-mimetic activity. The catalyst exhibits thermophilic property, and maintains oxidative activity up to 75 °C, which allows the catalyzed degradation of the as-dissolved native lignin with high efficiency even without assistance of the electron mediator. In contrast, at this condition, the native copper-dependent oxidase completely lost its activity. This catalyst with superior stability and activity offer promise for sustainable lignin valorization through biocatalytic routes compatible with ionic liquid pretreatment, addressing limitations in native enzymes for industrially relevant conditions.

Exceptional Electron-Rich Heteroaromatic Pentacycle for Ultralow Band Gap Conjugated Polymers and Photothermal Therapy.

Stable redox-active conjugated molecules with exceptional electron-donating abilities are key components for the design and synthesis of ultralow band gap conjugated polymers. While hallmark electron-rich examples such as pentacene derivatives have been thoroughly explored, their poor air stability has hampered their broad incorporation into conjugated polymers for practical applications. Herein, we describe the synthesis of the electron-rich, fused pentacyclic pyrazino[2,3-b:5,6-b']diindolizine (PDIz) motif and detail its optical and redox behavior. The PDIz ring system exhibits a lower oxidation potential and a reduced optical band gap than the isoelectronic pentacene while retaining greater air stability in both solution and the solid state. The enhanced stability and electron density, together with readily installed solubilizing groups and polymerization handles, allow for the use of the PDIz motif in the synthesis of a series of conjugated polymers with band gaps as small as 0.71 eV. The tunable absorbance throughout the biologically relevant near-infrared I and II regions enables the use of these PDIz-based polymers as efficient photothermal therapeutic reagents for laser ablation of cancer cells.

Binding of Proteins to Copolymers of Varying Charges and Hydrophobicity: A Molecular Mechanism and Computational Strategies.

Because of their ability to selectively bind to a target protein, copolymer nanoparticles (NPs) containing a selected combination of hydrophobic and charged groups have been frequently reported as potent antibody-like analogues. However, due to the intrinsic disorder of the copolymer NP in terms of its random monomer sequence and the cross-linked copolymer matrix, the copolymer NP is indeed strikingly different from a well-folded protein antibody and the complexation between the copolymer NP and a target protein is likely not due to a lock-key type of interaction but possibly due to a novel and unexplored molecular mechanism. Here, we study a key biomarker protein, vimentin, interacting with a set of random copolymer chains using implicit-water explicit-ion coarse-grained (CG) molecular dynamics (MD) simulations along with biolayer interferometry (BLI) analysis. Due to the charge and hydrophobicity anisotropy on the vimentin dimer (VD) surface, a set of bound copolymers are found inhomogenously adsorbed on the VD, with energetic heterogeneity for different binding sites and cooperative effect in the adsorption. Increasing the charge or hydrophobicity of the copolymer may have different consequences on the adsorption. In this study, we found that with more copolymer charges, the protein coverage increases for copolymers of low hydrophobicity and decreases of high hydrophobicity, which is explained by the distribution and size of various functional patches on the VD in loading those copolymers. Employing a coverage-dependent Langmuir model, we propose a simulation protocol to address the full profile of the copolymer binding free energy through the fit to the simulated binding isotherm. The obtained results correlate well with those from the BLI experiment, indicating the significance of this method for the rational design of the copolymer NP with engineered protein binding affinity.

Molecularly imprinted monoliths: Recent advances in the selective recognition of biomacromolecules related biomarkers.

Biomarkers are significant indicators to assist the early diagnosis of diseases and assess the therapeutic response. However, due to the low abundance of biomarkers in complex biological fluids, it is highly desirable to explore efficient techniques to attain their selective recognition and capture before the detection. Molecularly imprinted monoliths integrate the high selectivity of imprinted polymers and the rapid convective mass transport of monoliths, and as a result, are promising candidates to achieve the specific enrichment of biomarkers from complex samples. This review summarizes the various imprinting approaches for the preparation of molecularly imprinted monoliths. The state-of-art advances as an effective platform for applications in the selective capture of biomacromolecules related biomarkers were also outlined.

Selective recognition of tumor cells by molecularly imprinted polymers.

Molecularly imprinted polymers, developed 50 years ago, have garnered enormous attention as receptor-like materials. Lately, molecularly imprinted polymers have been employed as a specific target tool in favor of cancer diagnosis and therapy by the selective recognition of tumor cells. Although the molecular imprinting technology has been well-innovated recently, the cell still remains the most challenging target for imprinting. In this review, we summarize the advances in the synthesis of molecularly imprinted polymers suitable for the selective recognition of tumor cells. Through a sustained effort, three strategies have been developed including peptide-imprinting, polysaccharide-imprinting, and whole-cell imprinting, which have resulted in inspiring applications in effective cancer diagnosis and therapy. The major challenges and perspectives on the further directions related to the synthesis of molecularly imprinted polymers were also outlined.

Hierarchical Micro- and Mesoporous Zn-Based Metal-Organic Frameworks Templated by Hydrogels: Their Use for Enzyme Immobilization and Catalysis of Knoevenagel Reaction.

Encapsulation of enzymes in metal-organic frameworks (MOFs) is often obstructed by the small size of the orifices typical of most reported MOFs, which prevent the passage of larger-size enzymes. Here, the preparation of hierarchical micro- and mesoporous Zn-based MOFs via the templated emulsification method using hydrogels as a template is presented. Zinc-based hydrogels featuring a 3D interconnecting network are first produced via the formation of hydrogen bonds between melamine and salicylic acid in which zinc ions are well distributed. Further coordination with organic linkers followed by the removal of the hydrogel template produces hierarchical Zn-based MOFs containing both micropores and mesopores. These new MOFs are used for the encapsulation of glucose oxidase and horseradish peroxidase to prove the concept. The immobilized enzymes exhibit a remarkably enhanced increased operational stability and enzymatic activity with a kcat /km value of 85.68 mm s-1 . This value is 7.7-fold higher compared to that found for the free enzymes in solution, and 2.7-fold higher than enzymes adsorbed on conventional microporous MOFs. The much higher catalytic activity of the mesoporous conjugate for Knoevenagel reactions is demonstrated, since the large pores enable easier access to the active sites, and compared with that observed for catalysis using microporous MOFs.

Thermoresponsive hydrophobic copolymer grafted on agar microspheres for all-aqueous bioseparations.

Agar microspheres were prepared by water-oil emulsification and cross-linked under alkaline condition. The thermoresponsive hydrophobic copolymer, poly(N-isopropylacrylamide-co-lauryl methacrylate-co-acrylamide), was grafted on the agar microspheres via atom transfer radical polymerization. The agar microspheres grafted with copolymers were characterized by light microphotography, elemental analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The chain lengths and hydrophobic monomer ratio of the grafting linear polymer had significant effects on the hydrophobicity and adsorption capacity of agar microspheres at different temperatures. The thermoresponsive microspheres were used for separation of proteins and showed binding and release behavior by change of temperatures without change in mobile phase composition. Thus, we suggest thermoresponsive agar microspheres as an alternative separation media for all-aqueous bioseparations.

Comparison of commercial organic polymer-based and silica-based monolithic columns using mixtures of analytes differing in size and chemistry.

Commercially available silica-based monolithic columns Chromolith RP-8e, Chromolith RP-18, and Chromolith HR RP-18, and polymer-based monolithic columns ProSwift RP-1S, ProSwift RP-2H, and ProSwift RP-3U varying in pore size and bonded phase have been tested for the fast separation of selected sets of analytes. These mixtures of analytes included small molecules (uracil, caffeine, 1-phenylethanol, butyl paraben, and anthracene), acylated insulins, and intact proteins (ribonuclease A, cytochrome C, transferrin, apomyoglobin, and thyroglobulin), and covered wide range of chemistries and sizes. Small molecules were well separated with a height equivalent to theoretical plate of 11-26 µm using silica-based monolithic columns, while organic polymer-based monoliths excelled in the fast sub 1 min baseline separations of large molecules. A peak capacity of 37 was found for separation of acylated insulins on Chromolith columns using a 3 min gradient at a flow rate of 3 ml/min. Poor recovery of proteins from Chromolith columns and significant peak tailing of small molecules using ProSwift columns were the major obstacles in using monolithic columns in those applications.

Preparation and applications of monolithic structures containing metal-organic frameworks.

Metal-organic frameworks are a new category of advanced porous materials with large surface areas and porosities, uniform pore sizes, tunable surface chemistry, and structural diversity. In combination with monoliths, they allow the fine tuning of desired interactions required in a variety of applications. This review article summarizes results of recent studies focused on synthetic strategies enabling incorporation of metal-organic frameworks in monolithic structures. A diverse array of applications including chromatographic separation, solid-phase microextraction, sample enrichment, heterogeneous catalysis, and enzymatic catalysis are also described.

Layer-by-Layer Assembly of Metal-Organic Frameworks in Macroporous Polymer Monolith and Their Use for Enzyme Immobilization.

New monolithic materials comprising zeolitic imidazolate framework (ZIF-8) located on the pore surface of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith previously functionalized with N-(3-aminopropyl)-imidazole have been prepared via a layer-by-layer self-assembly strategy. These new ZIF-8@monolith hybrids are used as solid-phase carriers for enzyme immobilization. Their performance is demonstrated with immobilization of a model proteolytic enzyme trypsin. The best of the conjugates enable very efficient digestion of proteins that can be achieved in mere 43 s.

"Smart" molecularly imprinted monoliths for the selective capture and easy release of proteins.

A new thermally switchable molecularly imprinted monolith for the selective capture and release of proteins has been designed. First, a generic poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith reacted with ethylenediamine followed by functionalization with 2-bromoisobutyryl bromide to introduce the initiator for atom transfer radical polymerization. Subsequently, a protein-imprinted poly(N-isopropylacrylamide) layer was grafted onto the surface of the monolithic matrix by atom transfer radical polymerization. Scanning electron microscopy and energy-dispersive X-ray spectroscopy of the cross-sections of imprinted monoliths confirmed the formation of dense poly(N-isopropylacrylamide) brushes on the pore surface. The imprinted monolith exhibited high specificity and selectivity toward its template protein myoglobin over competing proteins and a remarkably large maximum adsorption capacity of 1641 mg/g. Moreover, this "smart" imprinted monolith featured thermally responsive characteristics that enabled selective capture and easy release of proteins triggered only by change in temperature with water as the mobile phase and avoided use of stronger organic solvents or change in ionic strength and pH.

Preparation of reusable bioreactors using reversible immobilization of enzyme on monolithic porous polymer support with attached gold nanoparticles.

Porcine lipase has been reversibly immobilized on a monolithic polymer support containing thiol functionalities prepared within confines of a fused silica capillary and functionalized with gold nanoparticles. Use of gold nanoparticles enabled rejuvenation of the activity of the deactivated reactor simply by stripping the inactive enzyme from the nanoparticles using 2-mercaptoethanol and subsequent immobilization of fresh lipase. This flow through enzymatic reactor was then used to catalyze the hydrolysis of glyceryl tributyrate (tributyrin). The highest activity was found within a temperature range of 37-40°C. The reaction kinetics is characterized by Michaelis-Menten constant, Km = 10.9 mmol/L, and maximum reaction rate, Vmax = 5.0 mmol/L min. The maximum reaction rate for the immobilized enzyme is 1,000 times faster compared to lipase in solution. The fast reaction rate enabled to achieve 86.7% conversion of tributyrin in mere 2.5 min and an almost complete conversion in 10 min. The reactor lost only less than 10% of its activity even after continuous pumping through it a solution of substrate equaling 1,760 reactor volumes. Finally, potential application of this enzymatic reactor was demonstrated with the transesterification of triacylglycerides from kitchen oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.

New agar microspheres for the separation and purification of natural products.

A new type of agar chromatography media has been prepared with a yield over 80% using a water-in-oil emulsion technique. These microspheres have regular spherical shapes and particle diameters in the range 40-165 µm (average ∼90 µm). Cross-linking of the resulting agar microspheres with epichlorohydrin and 1,4-butanediol diglycidyl ether enhanced their mechanical and thermal stability. The alkaline conditions used during the cross-linking reaction also decreased the content of ionized sulfate groups of the polysaccharide, thus reducing the nonspecific adsorption of positively charged molecules. The cross-linked agar microspheres were functionalized with (i) branched poly(ethyleneimine) to obtain a stationary phase useful for the separation of proteins in an anion-exchange mode and (ii) with poly-ß-cyclodextrin enabling direct isolation and purification of puerarin from a crude extract of Radix puerariae. Using a 23.5 mL column loaded with 20 mg extract (0.85 mg/mL gel), puerarin with a purity of 96% was recovered with a yield of 86%.

Boosted Enzyme Activity via Encapsulation within Metal-Organic Frameworks with Pores Matching Enzyme Size and Shape.

A novel and versatile approach called "physical imprinting" is introduced to modulate enzyme conformation using mesoporous materials, addressing challenges in achieving improved enzyme activity and stability. Metal-organic frameworks with tailored mesopores, precisely matching enzyme size and shape, are synthesized. Remarkably, enzymes encapsulated within these customized mesopores exhibit over 1670% relative activity compared to free enzymes, maintaining outstanding efficiency even under harsh conditions such as heat, exposure to organic solvents, wide-ranging pH extremes from acidic to alkaline, and exposure to a digestion cocktail. After 18 consecutive cycles of use, the immobilized enzymes retain 80% of their initial activity. Additionally, the encapsulated enzymes exhibit a substantial increase in catalytic efficiency, with a 14.1-fold enhancement in kcat/KM compared to native enzymes. This enhancement is among the highest reported for immobilized enzymes. The improved enzyme activity and stability are corroborated by solid-state UV-vis, electron paramagnetic resonance, Fourier-transform infrared spectroscopy, and solid-state NMR spectroscopy. The findings not only offer valuable insights into the crucial role of size and shape complementarity within confined microenvironments but also establish a new pathway for developing solid carriers capable of enhancing enzyme activity and stability.

Synthetic Polymer Nanoparticles as an Abiotic Artificial Inhibitor of Tyrosinase.

An innovative methodology is presented for synthesizing synthetic polymer nanoparticles (TINPs) as potent tyrosinase inhibitors. This inhibition strategy combines the integration of two distinct functionalities, phenol, and phenylboronic acid, within the TINPs structure. The phenyl group mimics the natural monophenol substrate, forming a strong coordination with the catalytic copper ion, significantly inhibiting tyrosinase activity. Additionally, phenylboronic acid interacts with catechol, another tyrosinase substrate, further reducing enzyme efficiency. The shared benzene ring in phenyl and phenylboronic acid enhances binding to tyrosinase's hydrophobic pocket near its copper active site, contributing to potent inhibition. TINPs exhibit exceptional performance, boasting an impressive IC50 value of 3.5×10-8 m and an inhibition constant of 9.8×10-9 m. Validation of the approach is unequivocally demonstrated through the successful inhibition of tyrosinase activity and melanin production, substantiated in both in vitro and in vivo scenarios. The mechanism of TINP inhibition is elucidated through circular dichroism and Fourier transform infrared spectroscopy. This study introduces a versatile design approach for developing abiotic polymer-based enzyme inhibitors, expanding possibilities in enzyme inhibition research.

Transgenic rhesus monkeys produced by gene transfer into early-cleavage-stage embryos using a simian immunodeficiency virus-based vector.

The development of transgenic technologies in monkeys is important for creating valuable animal models of human physiology so that the etiology of diseases can be studied and potential therapies for their amelioration may be developed. However, the efficiency of producing transgenic primate animals is presently very low, and there are few reports of success. We have developed an improved methodology for the production of transgenic rhesus monkeys, making use of a simian immunodeficiency virus (SIV)-based vector that encodes EGFP and a protocol for infection of early-cleavage-stage embryos. We show that infection does not alter embryo development. Moreover, the timing of infection, either before or during embryonic genome activation, has no observable effect on the level and stability of transgene expression. Of 70 embryos injected with concentrated virus at the one- to two-cell stage or the four- to eight-cell stage and showing fluorescence, 30 were transferred to surrogate mothers. One transgenic fetus was obtained from a fraternal triple pregnancy. Four infant monkeys were produced from four singleton pregnancies, of which two expressed EGFP throughout the whole body. These results demonstrate the usefulness of SIV-based lentiviral vectors for the generation of transgenic monkeys and improve the efficiency of transgenic technology in nonhuman primates.

Rapid Screening and Synthesis of Abiotic Synthetic Receptors for Selective Bacterial Recognition.

The major challenges that impede the preparation of abiotic synthetic receptors designed to feature selective bacterial recognition properties are the complexity, nonrobustness, and environmental adaptability of live microbes. Here, we describe a new rapid screening strategy to determine the optimal polymer formulation on 96-well plates and then produce abiotic synthetic receptors by imprinting the surface marker lipopolysaccharide (LPS) of Gram-negative bacteria. The resulting LPS-imprinted nanoparticles reveal remarkable affinity toward LPS with an equilibrium dissociation constant (KD) value of 10-12 M and can distinguish and selectively recognize specific bacteria in whole blood at concentrations down to 10 cells/mL. The incorporation of gold nanorods into imprinted nanoparticles allows selective microbial inactivation based on photothermal treatment. We have also demonstrated that the imprinted nanoparticles with high affinity for bacteria could induce bacteria clustering, drive the expression of quorum-sensing-controlled signal molecules, and eventually enhance the productivity of the cell factory.

Solid-Phase Screening and Synthesis of Molecularly Imprinted Nanoparticles for Selective Recognition and Detection of Brain Natriuretic Peptide.

A new recognition method is explored for the rapid detection of B-type natriuretic peptide (BNP) based on the rational design and solid-phase synthesis of molecularly imprinted nanoparticles (nanoMIP) encapsulated with carbon dots. The nanosized magnetic template is first prepared by attaching the epitope of BNP on amino-functionalized magnetic carriers. High-dilution polymerization of monomers in the presence of magnetic template generates lightly crosslinked imprinted nanoparticles. To obtain the optimal MIP formulation, a new combinatorial screening approach is developed by a competitive fluorescence assay using the magnetic template. The resultant nanoMIP exhibits high affinity and selectivity toward BNP with an equilibrium dissociation constant (KD ) of ≈10-11  m. The proposed assay allows fast BNP detection within ≈7 min with a linear range of its concentration from 0.25 to 5000 pg mL-1 and a limit of detection of 0.208 pg mL-1 (S/N = 3). To demonstrate its practicability in clinical diagnosis, unknown real serum samples from 160 individuals are analyzed and the relative standard deviation is less than 4.43%. Compared with the routine electrochemiluminescence detection method that is widely used in hospital, the relative error is less than 4.98% and the correlation coefficient is 0.994.