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.

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-1ß, 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.

Neutrophil Activation by Mineral Microparticles Coated with Methylglyoxal-Glycated Albumin.

Hyperglycemia-induced protein glycation and formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of diabetic complications and pathological biomineralization. Receptors for AGEs (RAGEs) mediate the generation of reactive oxygen species (ROS) via activation of NADPH-oxidase. It is conceivable that binding of glycated proteins with biomineral particles composed mainly of calcium carbonate and/or phosphate enhances their neutrophil-activating capacity and hence their proinflammatory properties. Our research managed to confirm this hypothesis. Human serum albumin (HSA) was glycated with methylglyoxal (MG), and HSA-MG was adsorbed onto mineral microparticles composed of calcium carbonate nanocrystals (vaterite polymorph, CC) or hydroxyapatite nanowires (CP). As scopoletin fluorescence has shown, H2O2 generation by neutrophils stimulated with HSA-MG was inhibited with diphenyleneiodonium chloride, wortmannin, genistein and EDTA, indicating a key role for NADPH-oxidase, protein tyrosine kinase, phosphatidylinositol 3-kinase and divalent ions (presumably Ca2+) in HSA-MG-induced neutrophil respiratory burst. Superoxide anion generation assessed by lucigenin-enhanced chemiluminescence (Luc-CL) was significantly enhanced by free HSA-MG and by both CC-HSA-MG and CP-HSA-MG microparticles. Comparing the concentrations of CC-bound and free HSA-MG, one could see that adsorption enhanced the neutrophil-activating capacity of HSA-MG.

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.

Cooling-Triggered Release from Mesoporous Poly(N-isopropylacrylamide) Microgels at Physiological Conditions.

Poly(N-isopropylacrylamide) (pNIPAM) hydrogels have broad potential applications as drug delivery vehicles because of their thermoresponsive behavior. pNIPAM loading/release performances are directly affected by the gel network structure. Therefore, there is a need with the approaches for accurate design of 3D pNIPAM assemblies with the structure ordered at the nanoscale. This study demonstrates size-selective spontaneous loading of macromolecules (dextrans 10-500 kDa) into pNIPAM microgels by microgel heating from 22 to 35 °C (microgels collapse and trap dextrans) followed by the dextran release upon further cooling down to 22 °C (microgels swell back) . This temperature-mediated behavior is fully reversible. The structure of pNIPAM microgels was tailored via hard templating and cross-linking of the hydrogel using sacrificial mesoporous cores of vaterite CaCO3 microcrystals. In addition, the fabrication of hollow thermoresponsive pNIPAM microshells has been demonstrated, utilizing vaterite microcrystals that had narrower pores. The proposed approach for heating-triggered encapsulation and cooling-triggered release into/from pNIPAM microgels may pave the ways for applications of pNIPAM hydrogels for skin and transdermal cooling-responsive drug delivery in the future.

Inter-protein interactions govern protein loading into porous vaterite CaCO3 crystals.

The fast development of protein therapeutics has resulted in a high demand for advanced delivery carriers that can effectively host therapeutic proteins, preserve their bioactivity and release them on demand. Accordingly, vaterite CaCO3 crystals have attracted special attention as sacrificial templates for protein encapsulation in micro- and nanoparticles (capsules and beads, respectively) under mild biofriendly conditions. This study aimed to better understand the mechanism of protein loading into crystals as a primary step for protein encapsulation. The loading of three therapeutic proteins (250 kDa catalase, 5.8 kDa insulin, and 6.5 kDa aprotinin) was investigated for crystals with different porosities. However, unexpectedly, the protein loading capacity was not consistent with the protein molecular weight. It solely depends on the inter-protein interactions in the bulk solution in the presence of crystals and that inside the crystals. The smallest protein aprotinin aggregates in the bulk (its aggregate size is about 100 nm), which prohibits its loading into the crystals. Insulin forms hexamers in the bulk, which can diffuse into the crystal pores but tend to aggregate inside the pores, suppressing protein diffusion inward. Catalase, the largest protein tested, does not form any aggregates in the bulk and diffuses freely into the crystals; however, its diffusion into small pores is sterically restricted. These findings are essential for the encapsulation of protein therapeutics by means of templating based on CaCO3 crystals and for the engineering of protein-containing microparticles having desired architectures.

Bio-friendly encapsulation of superoxide dismutase into vaterite CaCO3 crystals. Enzyme activity, release mechanism, and perspectives for ophthalmology.

Mesoporous vaterite CaCO3 crystals are nowadays one of the most popular vectors for loading of fragile biomolecules like proteins due to biocompatibility, high loading capacity, cost effective and simple loading procedures. However, recent studies reported the reduction of bioactivity for protein encapsulation into the crystals in water due to rather high alkaline pH of about 10.3 caused by the crystal hydrolysis. In this study we have investigated how to retain the bioactivity and control the release rate of the enzyme superoxide dismutase (SOD) loaded into the crystals via co-synthesis. SOD is widely used as an antioxidant in ophthalmology and its formulations with high protein content and activity as well as opportunities for a sustained release are highly desirable. Here we demonstrate that SOD co-synthesis can be done at pH 8.5 in a buffer without affecting crystal morphology. The synthesis in the buffer allows reaching the high loading efficiency of 93%, high SOD content (24 versus 15 w/w % for the synthesis in water), and order of magnitude higher activity compared to the synthesis in water. The enormous SOD concentration into crystals of 10-2 M is caused by the entrapment of SOD aggregates into the crystal pores. The SOD released from crystals at physiologically relevant ionic strength fully retains its bioactivity. As found by fitting the release profiles using zero-order and Baker-Lonsdale models, the SOD release mechanism is governed by both the SOD aggregate dissolution and by the diffusion of SOD molecules thorough the crystal pores. The latest process contributes more in case of the co-synthesis in the buffer because at higher pH (co-synthesis in water) the unfolded SOD molecules aggregate stronger. The release is bi-modal with a burst (ca 30%) followed by a sustained release and a complete release due to the recrystallization of vaterite crystals to non-porous calcite crystals. The mechanism of SOD loading into and release from the crystals as well as perspectives for the use of the crystals for SOD delivery in ophthalmology are discussed. We believe that together with a fundamental understanding of the vaterite-based protein encapsulation and protein release, this study will help to establish a power platform for a mild and effective encapsulation of fragile biomolecules like proteins at bio-friendly conditions.

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.

Extremely high-frequency electromagnetic radiation enhances neutrophil response to particulate agonists.

The growing use of extremely high-frequency electromagnetic radiation (EHF EMR) in information and communication technology and in biomedical applications has raised concerns regarding the potential biological impact of millimeter waves (MMWs). Here, we elucidated the effects of MMW radiation on neutrophil activation induced by opsonized zymosan or E. coli in whole blood ex vivo. After agonist addition to blood, two samples were prepared. A control sample was incubated at ambient conditions without any treatment, and a test sample was exposed to EHF EMR (32.9-39.6 GHz, 100 W/m2 ). We used methods that allowed us to assess the functional status of neutrophils immediately after exposure: oxidant production levels were measured by luminol-dependent chemiluminescence, and morphofunctional changes to neutrophils were observed in blood smears. Results revealed that the response of neutrophils to both agonists was intensified if blood was exposed to MMW radiation for 15 min. Neutrophils were intact in both the control and irradiated samples if no agonist was added to blood before incubation. Similarly, exposing suspensions of isolated neutrophils in plasma to MMW radiation enhanced cell response to both zymosan and E. coli. Heating blood samples was shown to be the primary mechanism underlying enhanced EHF EMR-induced oxidant production by neutrophils in response to particulate agonists. Bioelectromagnetics. 39:144-155, 2018. © 2017 Wiley Periodicals, Inc.

Protein-Containing Multilayer Capsules by Templating on Mesoporous CaCO3 Particles: POST- and PRE-Loading Approaches.

Encapsulation of model proteins (catalase, insulin, aprotinin) into multilayer dextran sulphate/protamin capsules by templating on CaCO3 microparticles is investigated employing: (i) PRE-loading into CaCO3 particles by adsorption or co-synthesis and (ii) POST-loading into performed capsules. Protein encapsulation is governed by both its size and electrostatic interactions with the carbonate microparticles and multilayer shell. PRE-loading enables improved encapsulation compared to POST-loading (catalase content in capsules 630 and 70 mg · g(-1)). Bioactivity of encapsulated protein is not affected by interaction with multilayers but may be reduced at slightly alkaline pH due to CaCO3 hydrolysis. This study might help to successfully encapsulate fragile bio-macromolecules into multilayer capsules.

Multifunctional polyelectrolyte microparticles for oral insulin delivery.

Multicomponent insulin-containing microparticles are prepared by layer-by-layer assembly of dextran sulfate and chitosan on the core of protein-polyanion complex with or without protease inhibitors. Oral bioavailability of the encapsulated insulin is improved due to the cumulative effect of each component. A physico-chemical study shows that the particle design allows adjustment of the pH-dependent profile of the insulin release, as well as mucoadhesive properties and Ca(2+) binding ability of the microparticles. Supplementing the microparticles with 2-3% protease inhibitors fully prevents proteolysis of human insulin. The pharmacological effect of microencapsulated insulin in doses 50-100 IU kg(-1) is demonstrated in chronic experiments after oral administration to diabetic rats fed ad libitum.