A High-Throughput Screening Strategy for Synthesizing Molecularly Imprinted Polymer Nanoparticles Selectively Targeting Tumors.

Molecularly imprinted polymers (MIPs) show significant promise as effective alternatives to antibodies in disease diagnosis and therapy. However, the challenging process of screening extensive libraries of monomer combinations and synthesis conditions to identify formulations with enhanced selectivity and affinity presents a notable time constraint. The need for expedient methods becomes clear in accelerating the strategic development of MIPs tailored for precise molecular recognition purposes. In this study, an innovative high-throughput screening methodology designed to identify the optimal MIP formulation for targeting tumors is presented. Employing a microtiter plate format, over 100 polymer syntheses are conducted, incorporating diverse combinations of functional monomers. Evaluation of binding performance utilizes fluorescence-based assays, focusing on an epitope of the epidermal growth factor receptor (EGFR). Through this meticulously structured screening process, synthesis conditions that produced MIP nanoparticles exhibiting substantial specificity for EGFR targeting (KD = 10-12 m) are identified. These "bionic antibodies" demonstrate selective recognition of cancer cells in whole blood samples, even at concentrations as low as 5 cells mL-1. Further validation through fluorescent imaging confirms the tumor-specific localization of the MIPs in vivo. This highly efficient screening approach facilitates the strategic synthesis of imprinted polymers functioning as precision bioprobes.

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.

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.

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.

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.

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.

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.

Isometric Covalent Triazine Framework-Derived Porous Carbons as Metal-Free Electrocatalysts for the Oxygen Reduction Reaction.

Covalent triazine frameworks (CTFs) and their derivative N-doped carbons have attracted much attention for application in energy conversion and storage. However, previous studies have mainly focused on developing new building blocks and optimizing synthetic conditions. The use of isometric building blocks to control the porous structure and to fundamentally understand structure-property relationships have rarely been reported. In this work, two isometric building blocks are used to produce isometric CTFs with controllable pore geometries. The as-prepared CTF with nonplanar hexagonal rings demonstrates higher surface area, larger pore volume, and richer N content than the planar CTF. After pyrolysis, nonplanar porous CTF-derived N-doped carbons exhibit admirable catalytic activity for oxygen reduction in alkaline media (half-wave potential: 0.86 V; Tafel slope: 65 mV dec-1 ), owing to their larger pore volume and the abundance of pyridinic and graphitic N species. When assembled into a zinc-air battery, the as-made electrocatalysts show high capacities of up to 651 mAh g-1 and excellent durability.

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.

Synthesis and characterization of polymerizable MOFs for the preparation of MOF/polymer mixed matrix membranes.

Mixed-matrix membranes (MMMs) comprising polymer matrices and metal-organic frameworks (MOFs) provide efficient and economic CO2 separation. One major challenge is to construct continuous and defect-free MMMs due to poor MOF/polymer compatibility. Here, this protocol describes the step-by-step details for synthesis of desired linkers that allow the fabrication of new polymerizable MOFs containing vinyl groups (BUCT MOFs) and the preparation procedures of defect-free MMMs with enhanced MOF/polymer interfacial adhesion and boosted gas separation performances. For complete details on the use and execution of this profile, please refer to Chen et al. (2021).

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.

Hybrid Porous Crystalline Materials from Metal Organic Frameworks and Covalent Organic Frameworks.

Two frontier crystalline porous framework materials, namely, metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), have been widely explored owing to their outstanding physicochemical properties. While each type of framework has its own intrinsic advantages and shortcomings for specific applications, combining the complementary properties of the two materials allows the engineering of new classes of hybrid porous crystalline materials with properties superior to the individual components. Since the first report of MOF/COF hybrid in 2016, it has rapidly evolved as a novel platform for diverse applications. The state-of-art advances in the various synthetic approaches of MOF/COF hybrids are hereby summarized, together with their applications in different areas. Perspectives on the main challenges and future opportunities are also offered in order to inspire a multidisciplinary effort toward the further development of chemically diverse, multi-functional hybrid porous crystalline materials.

Boosted activity by engineering the enzyme microenvironment in cascade reaction: A molecular understanding.

Engineering of enzyme microenvironment can surprisingly boost the apparent activity. However, the underlying regulation mechanism is not well-studied at a molecular level so far. Here, we present a modulation of two model enzymes of cytochrome c (Cty C) and d-amino acid oxidase (DAAO) with opposite pH-activity profiles using ionic polymers. The operational pH of poly (acrylic acid) modified Cyt C and polyallylamine modified DAAO was extended to 3-7 and 2-10 where the enzyme activity was larger than that at their optimum pH of 4.5 and 8.5 by 106% and 28%, respectively. The cascade reaction catalyzed by two modified enzymes reveals a 1.37-fold enhancement in catalytic efficiency compared with their native counterparts. The enzyme activity boosting is understood by performing the UV-vis/CD spectroscopy and molecular dynamics simulations in the atomistic level. The increased activity is ascribed to the favorable microenvironment in support of preserving enzyme native structures nearby cofactor under external perturbations.

Abiotic Mimic of Matrix Metalloproteinase-9 Inhibitor against Advanced Metastatic Cancer.

As the most representative family of proteinases related to tumorigenesis, matrix metalloproteinase-9 (MMP-9) represents a key player in cancer cell migration and regulation of the tumor microenvironment. The inhibition of MMP-9 activity has been pursued as a target for anticancer therapy. However, most synthetic MMP-9 inhibitors have failed in clinical trials because of their lack of selectivity. Here, an abiotic mimic based on molecularly imprinted nanoparticles has been designed as an inhibitor for MMP-9. To attain fast mass transfer and facilitate multifunctional roles, we synthesized the imprinted polymer thin layer on the surface of gold nanorods by reversible addition-fragmentation chain transfer polymerization using MMP-9 as the template, which captures MMP-9 selectively and inhibits its activity by providing steric hindrance to the activity-related domain of MMP-9. In vitro cell experiments and in vivo studies in mice demonstrate that the imprinted artificial antibody suppresses the migration and growth of metastatic tumors. The tumor growth inhibition rate reaches up to 54 ± 15%. Compared with the typical photothermal therapy induced by gold nanorods, the use of MMP-9-imprinted synthetic antibody could better inhibit the lung tumor metastasis by quenching the enzyme activity of MMP-9. This study offers a new paradigm in the engineering of imprinted nanoparticles as inhibitors for cancer therapy.

Polymerizable metal-organic frameworks for the preparation of mixed matrix membranes with enhanced interfacial compatibility.

The preparation of flawless and defect-free mixed matrix membranes (MMMs) comprising metal-organic framework (MOF) and polymer is often difficult owing to the poor MOF/polymer interface compatibility. Herein, we present the synthesis of an important family of pillared-layered MOFs with polymerizable moieties based on the parent structure [Zn2L2P]n [L = vinyl containing benzenedicarboxylic acid linkers; P = 4,4'-bipyridine (bipy)]. The crystalline structures of polymerizable MOFs were analyzed using single-crystal X-ray crystallography. The presence of reactive double bonds in MOFs was verified by the successful thiol-ene click reaction with sulfhydryl compounds. The subsequent copolymerization of polymerizable MOFs with organic monomers produced mixed matrix membranes with enhanced MOF/polymer interfacial adhesion that enabled good separation efficiency of CO2 from flue gas. This strategy provides a stimulating platform to the preparation of highly efficient MMMs that are capable of mitigating energy consumption and environment issues.

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.

Molecularly imprinted polymers for the selective recognition of microorganisms.

Molecularly imprinted polymers (MIPs) emerged half a century ago have now attracted tremendous attention as artificial receptors or plastic antibodies. Although the preparation of MIPs targeting small molecules, peptides, or even proteins is straightforward and well-developed, the molecular imprinting of microorganisms still remains a big challenge. This review highlights the preparation of MIPs that reveal biomimetic specificity and selectivity towards microorganisms by creating the well-defined cell recognition sites. We present the state-of-the-art strategies for the expeditious synthesis of MIPs targeting microorganism including surface components imprinting, cell mediated lithography, and microcontact stamping. These receptor-like biomimetic materials have garnered increasing attention in different fields. In this review, we also describe the diverse applications of microorganism-imprinted polymers such as microbial activation, microbial fuel cells, and microorganism detection and sensing. The major challenges and further prospects on the design of microorganism-imprinted polymers is also outlined.