Enhanced Activity and Stability of Lysozyme by Immobilization in the Matching Nanochannels of Mesoporous Silica Nanoparticles

Abstract

It is highly desirable to study the kinetics and spectroscopy of enzymes in a crowded and controllable microenvironment. In this work, we employ mesoporous silica of matching pore sizes to confine lysozyme in order to mimic enzyme in a crowded environment. The stability and activity of lysozyme immobilized in mesoporous silica nanoparticle (MSN) of various pore sizes were studied and correlated to spectroscopic data of the immobilized enzyme. By siteselective surface functionalization, we were able to avoid protein adsorbing on the external surfaces of MSNs and study specifically the protein immobilized in the nanochannels. Solution spectroscopic methods, CD and fluorescence, were used to study the secondary and tertiary structures of the immobilized enzyme because MSNs could be suspended very well in solution. To study the catalytic activity of lysozyme, we employed 4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotrioside as a substrate that was hydrolyzed and detected by fluorescence spectroscopy. 8-Anilino-1-naphthalenesulfonic acid was utilized as a fluorescence probe to characterize the protein-binding site. The conformation, thermal stability, and catalytic activity of lysozyme were sensitive to the curvature of the silica materials. The activity of the lysozyme immobilized in the 5.6 nm mesopores of MSNs was higher than those of native enzymes. The enhanced activity was attributed to subtle change in tertiary structure of lysozyme in the crowded microenvironment in the mesopores.

Controlled release and intracellular protein delivery from mesoporous silica nanoparticles

“…It was found that the confinement of lysozyme within 5.6 nm pores limited unfolding and hence increased the thermal stability of the protein. Additionally, this study showed superior catalytic activity by enzymes within the nanochannels as compared to those in the cytoplasm [58].…”

“…Kao et al devised a scheme to intentionally prevent this via opposing electrostatic interactions with site-selective surface functionalization, which allowed them to load lysozyme exclusively in the nanochannels [58]. Mesoporous silica nanoparticles were first functionalized with 3-aminopropyltriethoxysilane (APTES) such that the amines on the external surfaces became positively charged in a buffer solution at pH 3.2, giving the MSN surface a net positive charge.…”

Colloidal Mesoporous Silica Nanoparticles

“…In fact, the surfaces of colloidal MSNs have been successfully modified with the retention of their dispersibility by using various hydrophilic silylating agents, such as PEG-silane and amino- propyltriethoxysilane. 98,128,129,131,132,134,135 …”

“…54,170 In general, MSNs have been modified chemically to introduce functions, such as controlled release of drugs and catalysis. 135,171,172 For example, Nel et al prepared MSNs functionalized with supramolecular nanovalves that are tightly closed at physiological pH (7.4) but capable of opening and delivering drugs in acidifying cellular compartments. The β-cyclodextrin ring encircles the aromatic amines owing to their noncovalent bonding interactions under neutral pH conditions, and effectively blocks the opening of the pores and traps introduced drugs (Figure 17).…”

Template-based syntheses for shape controlled nanostructures

“…Instead, a trend toward larger pore MSNs, HMSNs, and other pore-expanded MSNs that are capable of delivering larger compounds for genetic or peptide therapy applications have been shown to be more effective in targeting cancer resistant cells [462]. With pore expansion techniques, as those mentioned earlier with TMB, delivery of larger proteins [383,[463][464][465] and even genetic material [207,391] are possible.…”