Incorporation of Pectin into Vaterite Microparticles Prevented Effects of Adsorbed Mucin on Neutrophil Activation.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP-luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

In Situ SERS Sensing by a Laser-Induced Aggregation of Silver Nanoparticles Templated on a Thermoresponsive Polymer.

A stimuli-responsive (pH- and thermoresponsive) micelle-forming diblock copolymer, poly(1,2-butadiene)290-block-poly(N,N-dimethylaminoethyl methacrylate)240 (PB-b-PDMAEMA), was used as a polymer template for the in situ synthesis of silver nanoparticles (AgNPs) through Ag+ complexation with PDMAEMA blocks, followed by the reduction of the bound Ag+ with sodium borohydride. A successful synthesis of the AgNPs on a PB-b-PDMAEMA micellar template was confirmed by means of UV-Vis spectroscopy and transmission electron microscopy, wherein the shape and size of the AgNPs were determined. A phase transition of the polymer matrix in the AgNPs/PB-b-PDMAEMA metallopolymer hybrids, which results from a collapse and aggregation of PDMAEMA blocks, was manifested by changes in the transmittance of their aqueous solutions as a function of temperature. A SERS reporting probe, 4-mercaptophenylboronic acid (4-MPBA), was used to demonstrate a laser-induced enhancement of the SERS signal observed under constant laser irradiation. The local heating of the AgNPs/PB-b-PDMAEMA sample in the laser spot is thought to be responsible for the triggered SERS effect, which is caused by the approaching of AgNPs and the generation of "hot spots" under a thermo-induced collapse and the aggregation of the PDMAEMA blocks of the polymer matrix. The triggered SERS effect depends on the time of a laser exposure and on the concentration of 4-MPBA. Possible mechanisms of the laser-induced heating for the AgNPs/PB-b-PDMAEMA metallopolymer hybrids are discussed.

Hybrid Mucin-Vaterite Microspheres for Delivery of Proteolytic Enzyme Chymotrypsin.

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.

Mucin adsorption on vaterite CaCO3 microcrystals for the prediction of mucoadhesive properties.

Porous vaterite CaCO3 crystals are widely used as containers for drug loading and as sacrificial templates to assemble polymer-based nano- and micro-particles at mild conditions. Special attention is paid nowadays to mucosal delivery where the glycoprotein mucin plays a crucial role as a main component of a mucous. In this work mucoadhesive properties of vaterite crystals have been tested by investigation of mucin binding to the crystals as a function of (i) time, (ii) glycoprotein concentration, (iii) adsorption conditions and (iv) degree of mucin desialization. Mucin adsorption follows Bangham equation indicating that diffusion into crystal pores is the rate-limiting step. Mucin strongly binds to the crystals (ΔG = -35 ±â€¯4 kJ mol-1) via electrostatic and hydrophobic interactions forming a gel and thus giving the tremendous mucin mass content in the crystals of up to 16%. Despite strong intermolecular mucin-mucin interactions, pure mucin spheres formed after crystal dissolution are unstable. However, introduction of protamine, actively used for mucosal delivery, makes the spheres stable via additional electrostatic bonding. The results of this work indicate that the vaterite crystals are extremely promising carriers for mucosal drug delivery and for development of test-systems for the analysis of the mucoadhesion.

Self-Assembled Mucin-Containing Microcarriers via Hard Templating on CaCO3 Crystals.

Porous vaterite crystals of CaCO3 are extensively used for the fabrication of self-assembled polymer-based microparticles (capsules, beads, etc.) utilized for drug delivery and controlled release. The nature of the polymer used plays a crucial role and discovery of new perspective biopolymers is essential to assemble microparticles with desired characteristics, such as biocompatibility, drug loading efficiency/capacity, release rate, and stability. Glycoprotein mucin is tested here as a good candidate to assemble the microparticles because of high charge due to sialic acids, mucoadhesive properties, and a tendency to self-assemble, forming gels. Mucin loading into the crystals via co-synthesis is twice as effective as via adsorption into preformed crystals. Desialylated mucin has weaker binding to the crystals most probably due to electrostatic interactions between sialic acids and calcium ions on the crystal surface. Improved loading of low-molecular-weight inhibitor aprotinin into the mucin-containing crystals is demonstrated. Multilayer capsules (mucin/protamine)3 have been made by the layer-by-layer self-assembly. Interestingly, the deposition of single mucin layers (mucin/water)3 has also been proven, however, the capsules were unstable, most probably due to additional (to hydrogen bonding) electrostatic interactions in the case of the two polymers used. Finally, approaches to load biologically-active compounds (BACs) into the mucin-containing microparticles are discussed.

A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers.

A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal (opt) conditions: pHopt 6.0-10.0, topt 20-30 °C, NaClopt 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.

Bacteriophage phi11 lysin: Physicochemical characterization and comparison with phage phi80α lysin.

Phage lytic enzymes are promising antimicrobial agents. Lysins of phages phi11 (LysPhi11) and phi80α (LysPhi80α) can lyse (destroy) cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The objectives of the study were to investigate the stability of lysins of phages phi11 and phi80α in storage and functioning conditions, to identify optimum storage conditions and causes of inactivation. Stability of the recombinant LysPhi11 and LysPhi80α was studied using turbidimetry. CD-spectroscopy, dynamic light scattering, and electrophoresis were used to identify causes of inactivation. At 37°C, pH 7.5 and concentration of NaCl not higher than 150mM, LysPhi11 molecules contain a high percentage of random coils (43%). However, in spite of this the enzyme has high activity (0.4-0.8OD600nms(-1)mg(-1)). In storage conditions (4°C and 22°C, pH 6.0-9.0, 10-500mM NaCl) LysPhi11 is inactivated by a monomolecular mechanism. The optimum storage conditions for LysPhi11 (4°C, pH 6.0-7.5, 10mM NaCl) were selected under which the time of the enzyme half-inactivation is 120-160 days. LysPhi80α stability is insufficient: at 37°C the enzyme loses half of its activity almost immediately; at 4°C and 22°C the time of half-inactivation of LysPhi80α varies in the range from several hours to 3 days. Despite the common properties in the manifestation of antistaphylococcal activity the kinetic behavior of the enzymes is different. LysPhi11 is a more promising candidate to be used as an antimicrobial agent.

Physicochemical characterization of the staphylolytic LysK enzyme in complexes with polycationic polymers as a potent antimicrobial.

Staphylococcus aureus causes many serious visceral, skin, and respiratory diseases. About 90% of its clinical strains are multi-drug resistant, but the use of bacteriophage lytic enzymes offers a viable alternative to antibiotic therapy. LysK, the phage K endolysin, can lyse S. aureus when purified and exposed externally. It has been investigated in its complexes with polycationic polymers (poly-l-lysines (PLLs) of molecular weights 2.5, 9.6, and 55.2 kDa and their block copolymers with polyethylene glycol PLL10-PEG114, PLL30-PEG114, and PLL30-PEG23) as a basis for creating active and stable antimicrobial. Complexing with polycationic PLLs produces a stabilizing effect on LysK due to structure ordering in its molecules and break-down of aggregates as a result of electrostatic interaction. The stability of LysK in the presence of PLL-PEG block copolymers improves by both electrostatic and hydrophobic mechanisms. Complexes of LysK with 2.5, 9.6, 55.2 kDa poly-l-lysines and PLL30-PEG114 have demonstrated sufficient stability at the temperatures of physiological activity (37 °C) and storage (4 °C and 22 °C).