Hybrid Mucin‐Vaterite Microspheres for Delivery of Proteolytic Enzyme Chymotrypsin

Abstract While the enteral delivery of proteolytic enzymes is widely established for combating many diseases as an alternative to antibiotic treatment, their local delivery only emerges as administration route enabling sustained release in a controlled manner on site. The latest requires the development of drug delivery systems suitable for encapsulation and preservation of enzymatic proteolytic activity. This study proposes hybrid microspheres made of mucin and biodegradable porous crystals of calcium carbonate (CC) as the carriers for chymotrypsin (CTR) delivery. CTR is impregnated into CC and hybrid CC/mucin (CCM) microspheres by means of sorption without any chemical modification. The loading of the CC with mucin enhances CTR retention on hybrid microspheres (adsorption capacity of ≈8.7 mg g−1 vs 4.7 mg g−1), recharging crystal surface due to the presence of mucin and diminishing the average pore diameter of the crystals from 25 to 8 nm. Mucin also retards recrystallization of vaterite into nonporous calcite improving stability of CCM microspheres upon storage. Proteolytic activity of CTR is preserved in both CC and CCM microspheres, being pH dependent. Temperature‐induced inactivation of CTR significantly diminishes by CTR encapsulation into CC and CCM microspheres. Altogether, these findings indicate promises of hybrid mucin‐vaterite microspheres for mucosal application of proteases

Immobilization of Antioxidant Enzyme Catalase on Porous Hybrid Microparticles of Vaterite with Mucin

Catalase is one of the crucial antioxidant enzymes with diverse applications in textile, food industries, wastewater treatment, cosmetics, and pharmaceutics, which, however, is highly sensitive to environmental challenges. Resisting the loss of activity and prolongation of formulation storage can be achieved via the catalase entrapment into insoluble carriers. Affordable and degradable vaterite is proposed as amicable material for catalase immobilization. To improve the carrier properties of the vaterite, it was co‐precipitated with mucin from the pig's stomach producing ca 5 μm hybrid mucin/vaterite microparticles. Catalase is impregnated into the crystals by means of adsorption without chemical modifications. The presence of mucin matrix partially hinders catalase penetration into the crystals and reduces the adsorption capacity (for 0.1 mg mL−1 catalase, ca 2.3 vs ca 1.5 mg g−1 for pristine and hybrid microparticles, respectively) but significantly promotes the protection of antioxidant activity upon storage and under the action of temperature, organic solvent (acetonitrile), and proteolytic enzyme (trypsin). Hybrid microcrystals are pH‐sensitive and better retain the enzyme at pH 3–5 due to catalase‐mucin complexation. Immobilized catalase can be used for 5–6 consecutive cycles until it loses catalytic activity. Altogether, these findings indicate promises of hybrid mucin/vaterite microparticles for immobilization of antioxidant enzymes

Activation of Neutrophils by Mucin–Vaterite Microparticles

Nano- and microparticles enter the body through the respiratory airways and the digestive system, or form as biominerals in the gall bladder, salivary glands, urinary bladder, kidney, or diabetic pancreas. Calcium, magnesium, and phosphate ions can precipitate from biological fluids in the presence of mucin as hybrid nanoparticles. Calcium carbonate nanocrystallites also trap mucin and are assembled into hybrid microparticles. Both mucin and calcium carbonate polymorphs (calcite, aragonite, and vaterite) are known to be components of such biominerals as gallstones which provoke inflammatory reactions. Our study was aimed at evaluation of neutrophil activation by hybrid vaterite–mucin microparticles (CCM). Vaterite microparticles (CC) and CCM were prepared under standard conditions. The diameter of CC and CCM was 3.3 ± 0.8 µm and 5.8 ± 0.7 µm, with ƺ-potentials of −1 ± 1 mV and −7 ± 1 mV, respectively. CC microparticles injured less than 2% of erythrocytes in 2 h at 1.5 mg mL−1, and no hemolysis was detected with CCM; this let us exclude direct damage of cellular membranes by microparticles. Activation of neutrophils was analyzed by luminol- and lucigenin-dependent chemiluminescence (Lum-CL and Luc-CL), by cytokine gene expression (IL-6, IL-8, IL-10) and release (IL-1β, IL-6, IL-8, IL-10, TNF-α), and by light microscopy of stained smears. There was a 10-fold and higher increase in the amplitude of Lum-CL and Luc-CL after stimulation of neutrophils with CCM relative to CC. Adsorption of mucin onto prefabricated CC microparticles also contributed to activation of neutrophil CL, unlike mucin adsorption onto yeast cell walls (zymosan); adsorbed mucin partially suppressed zymosan-stimulated production of oxidants by neutrophils. Preliminary treatment of CCM with 0.1–10 mM NaOCl decreased subsequent activation of Lum-CL and Luc-CL of neutrophils depending on the used NaOCl concentration, presumably because of the surface mucin oxidation. Based on the results of ELISA, incubation of neutrophils with CCM downregulated IL-6 production but upregulated that of IL-8. IL-6 and IL-8 gene expression in neutrophils was not affected by CC or CCM according to RT2-PCR data, which means that post-translational regulation was involved. Light microscopy revealed adhesion of CC and CCM microparticles onto the neutrophils; CCM increased neutrophil aggregation with a tendency to form neutrophil extracellular traps (NETs). We came to the conclusion that the main features of neutrophil reaction to mucin–vaterite hybrid microparticles are increased oxidant production, cell aggregation, and NET-like structure formation, but without significant cytokine release (except for IL-8). This effect of mucin is not anion-specific since particles of powdered kidney stone (mainly calcium oxalate) in the present study or calcium phosphate nanowires in our previous report also activated Lum-CL and Luc-CL response of neutrophils after mucin sorption