Poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) microspheres with a high copolymerized AA content were fabricated using rapid membrane emulsification technique. The uniform size, good hydrophilicity, and thermo sensitivity of the microspheres were favorable for trypsin immobilization. Trypsin molecules were immobilized onto the microspheres surfaces by covalent attachment. The effects of various parameters such as immobilization pH value, enzyme concentration, concentration of buffer solution, and immobilization time on protein loading amount and enzyme activity were systematically investigated. Under the optimum conditions, the protein loading was 493 6 20 mg g 21 and the activity yield of immobilized trypsin was 155% 6 3%. The maximum activity (V max ) and Michaelis constant (K m ) of immobilized enzyme were found to be 0.74 lM s 21 and 0.54 mM, respectively.The immobilized trypsin showed better thermal and storage stability than the free trypsin. The enzyme-immobilized microspheres with high protein loading amount still can show a thermo reversible phase transition behavior. The research could provide a strategy to immobilize enzyme for application in proteomics.

A novel strategy was developed to synthesize uniform semi-interpenetrating polymer network (semi-IPN) microspheres by premix membrane emulsification combined with one-step polymerization. Synthesized poly(acrylic acid) (PAAc) polymer chains were added prior to the inner water phase, which contained N-isopropylacrylamide (NIPAM) monomer, N,N 1 -methylene bisacrylamide (MBA) cross-linker, and ammonium persulfate (APS) initiator. The mixtures were pressed through a microporous membrane to form a uniform water-in-oil emulsion. By crosslinking the NIPAM in a PAAc-containing solution, microspheres with temperature-and pH-responsive properties were fabricated. The semi-IPN structure and morphology of the microspheres were confirmed by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The average diameter of the obtained microspheres was approximately 6.5 µm, with Span values of less than 1. Stimuli-responsive behaviors of the microspheres were studied by the cloud-point method. The results demonstrated that semi-IPN microspheres could respond independently to both pH and temperature changes. After storing in a PBS solution (pH 7.0) at 4˝C for 6 months, the semi-IPN microspheres remained stable without a change in morphology or particle size. This study demonstrated a promising method for controlling the synthesis of semi-IPN structure microspheres with a uniform size and multiple functionalities.

In recent years, Prussian blue nanoparticles (PBNPs), also named Prussian blue nano-enzymes, have been shown to demonstrate excellent multi-enzyme simulation activity and anti-inflammatory properties, and can be used as reactive oxygen scavengers. Their good biocompatibility and biodegradability mean that they are ideal candidates for in vivo use. PBNPs are highly efficient electron transporters with oxidation and reduction activities. PBNPs also show considerable promise as nano-drug carriers and biological detection sensors owing to their huge specific surface area, good chemical characteristics, and changeable qualities, which might considerably increase the therapeutic impact. More crucially, PBNPs, as therapeutic and diagnostic agents, have made significant advances in biological nanomedicine. This review begins with a brief description of the synthesis methods of PBNPs, then focuses on the applications of PBNPs in tissue regeneration and inflammation according to the different properties of PBNPs. This article will provide a timely reference for further study of PBNPs as therapeutic agents.