Hemolytic Activity and Cytotoxicity of Synthetic Nanoclays with Montmorillonite Structure for Medical Applications.

The factors influencing the appearance of toxicity in samples of synthetic montmorillonite with a systematically changing chemical composition Nax(Al, Mg)2-3Si4O10(OH)2 nH2O, which are potentially important for their use in medicine as drug carriers, targeted drug delivery systems, entero- and hemosorbents have been studied. Samples synthesized under hydrothermal conditions had the morphology of nanolayers self-organized into the nanosponge structures. The effect of the aluminum content, particle sizes, porosity, and ζ-potential of the samples on their toxicity was studied. The cytotoxic effect of the samples on eukaryotic cells Ea. hy 926 was determined using the MTT assay. The hemolytic activity of the samples in the wide concentration range in relation to human erythrocytes was also estimated. It has been established that the toxicity of aluminosilicate nanoparticles can be significantly reduced by correctly selecting their synthesis conditions and chemical composition, which opens up the opportunities for their use in medicine.

Particles Morphology Impact on Cytotoxicity, Hemolytic Activity and Sorption Properties of Porous Aluminosilicates of Kaolinite Group.

A comparative study of the properties of aluminosilicates of the kaolinite (Al2Si2O5(OH)4∙nH2O) group with different particles morphology has been carried out. Under conditions of directed hydrothermal synthesis, kaolinite nanoparticles with spherical, sponge, and platy morphologies were obtained. Raw nanotubular halloysite was used as particles with tubular morphology. The samples were studied by X-ray diffraction, SEM, solid-state NMR, low-temperature nitrogen adsorption, and the dependence of the zeta potential of the samples on the pH of the medium was defined. The sorption capacity with respect to cationic dye methylene blue in aqueous solutions was studied. It was found that sorption capacity depends on particles morphology and decreases in the series spheres-sponges-tubes-plates. The Langmuir, Freundlich, and Temkin models describe experimental methylene blue adsorption isotherms on aluminosilicates of the kaolinite subgroup with different particles morphology. To process the kinetic data, pseudo-first order and pseudo-second order were used. For the first time, studies of the dependence of hemolytic activity and cytotoxicity of aluminosilicate nanoparticles on their morphology were carried out. It was found that aluminosilicate nanosponges and spherical particles are not toxic to human erythrocytes and do not cause their destruction at sample concentrations from 0.1 to 1 mg/g. Based on the results of the MTT test, the concentration value that causes 50% inhibition of cell population growth (IC50, mg/mL) was calculated. For nanotubes, this value turned out to be the smallest-0.33 mg/mL. For samples with platy, spherical and nanosponge morphology, the IC50 values were 1.55, 2.68, and 4.69 mg/mL, respectively.

Aluminosilicate Nanosponges: Synthesis, Properties, and Application Prospects.

A simple one-step method is presented for fabricating inorganic nanosponges with a kaolinite [Al2Si2O5(OH)4] structure. The nanosponges were synthesized by the hydrothermal treatment of aluminosilicate gels in an acidic medium (pH = 2.6) at 220 °C without using organic cross-linking agents, such as cyclodextrin or polymers. The formation of the nanosponge morphology was confirmed by scanning electron microscopy, and the assignment of the synthesized aluminosilicates to the kaolinite group was confirmed by X-ray diffraction and infrared spectroscopy. The effect of the synthesis conditions, in particular, the nature (HCl, HF, NaOH, and H2O) and pH of the reaction medium (2.6, 7, and 12), as well as the duration of the synthesis (3, 6, and 12 days), on the morphology of aluminosilicates of the kaolinite group was studied. The sorption capacity of aluminosilicate nanosponges with respect to cationic (e.g., methylene blue) and anionic (e.g., azorubine) dyes in aqueous solutions was studied. The pH sensitivity of the surface ζ potential of the synthesized nanosponges was demonstrated. The dependence of the hemolytic activity (the ability to destroy erythrocytes) of aluminosilicate nanoparticles on the particle morphology (platy, spherical, and nanosponge) has been identified for the first time. Aluminosilicate nanosponges were not found to exhibit hemolytic activity. The prospects of using aluminosilicate nanosponges to prepare innovative functional materials for ecology and medicine applications, in particular, as matrices for drug delivery systems, were identified.

Comparison of the Antimicrobial and Hemolytic Activities of Various Forms of Silver (Ions, Nanoparticles, Bioconjugates) Stabilized in a Zeolite Matrix.

A quantitative and qualitative comparison of the antimicrobial and hemolytic activities of silver in various states, in the form of ions, nanoparticles, and bioconjugates with the antimicrobial protein lysozyme stabilized in an inert zeolite matrix, has been carried out. A synthetic zeolite with a ß structure was chosen as a zeolite matrix. Using the ion-exchange method, the method of chemical reduction, and treating the matrix with a silver hydrosol with specified characteristics, samples of zeolites with the same silver content in various forms (Ag+, Ag° - Ag°/Lyz) in the amounts of 0.8 and 5 wt % have been synthesized. The samples obtained were studied by a complex of physicochemical research methods: X-ray diffraction, UV absorption spectroscopy, low-temperature nitrogen adsorption, electron microscopy, and atomic absorption. Antimicrobial activity was assessed against antibiotic-resistant Gram-negative microbe (e.g., Escherichia coli ML-35, Pseudomonas aeruginosa 522/17 MDR, Klebsiella pneumoniae ESBL 344) and Gram-positive microbe (e.g., Staphylococcus aureus 1399/17). The hemolytic activity in relation to human erythrocytes was estimated. The results obtained showed significant antimicrobial activity with a simultaneously high hemolytic activity of ionic silver. Silver nanoparticles have a lower level of antimicrobial activity and toxicity. Bioconjugates of silver nanoparticles and lysozyme showed an optimal combination of antimicrobial properties and lack of hemolytic activity.

Silver Nanoparticles Functionalized With Antimicrobial Polypeptides: Benefits and Possible Pitfalls of a Novel Anti-infective Tool.

Silver nanoparticles (AgNPs) and antimicrobial peptides or proteins (AMPs/APs) are both considered as promising platforms for the development of novel therapeutic agents effective against the growing number of drug-resistant pathogens. The observed synergy of their antibacterial activity suggested the prospect of introducing antimicrobial peptides or small antimicrobial proteins into the gelatinized coating of AgNPs. Conjugates with protegrin-1, indolicidin, protamine, histones, and lysozyme were comparatively tested for their antibacterial properties and compared with unconjugated nanoparticles and antimicrobial polypeptides alone. Their toxic effects were similarly tested against both normal eukaryotic cells (human erythrocytes, peripheral blood mononuclear cells, neutrophils, and dermal fibroblasts) and tumor cells (human erythromyeloid leukemia K562 and human histiocytic lymphoma U937 cell lines). The AMPs/APs retained their ability to enhance the antibacterial activity of AgNPs against both Gram-positive and Gram-negative bacteria, including drug-resistant strains, when conjugated to the AgNP surface. The small, membranolytic protegrin-1 was the most efficient, suggesting that a short, rigid structure is not a limiting factor despite the constraints imposed by binding to the nanoparticle. Some of the conjugated AMPs/APs clearly affected the ability of nanoparticle to permeabilize the outer membrane of Escherichia coli, but none of the conjugated AgNPs acquired the capacity to permeabilize its cytoplasmic membrane, regardless of the membranolytic potency of the bound polypeptide. Low hemolytic activity was also found for all AgNP-AMP/AP conjugates, regardless of the hemolytic activity of the free polypeptides, making conjugation a promising strategy not only to enhance their antimicrobial potential but also to effectively reduce the toxicity of membranolytic AMPs. The observation that metabolic processes and O2 consumption in bacteria were efficiently inhibited by all forms of AgNPs is the most likely explanation for their rapid and bactericidal action. AMP-dependent properties in the activity pattern of various conjugates toward eukaryotic cells suggest that immunomodulatory, wound-healing, and other effects of the polypeptides are at least partially transferred to the nanoparticles, so that functionalization of AgNPs may have effects beyond just modulation of direct antibacterial activity. In addition, some conjugated nanoparticles are selectively toxic to tumor cells. However, caution is required as not all modulatory effects are necessarily beneficial to normal host cells.

Application of Antimicrobial Peptides of the Innate Immune System in Combination With Conventional Antibiotics-A Novel Way to Combat Antibiotic Resistance?

Rapidly growing resistance of pathogenic bacteria to conventional antibiotics leads to inefficiency of traditional approaches of countering infections and determines the urgent need for a search of fundamentally new anti-infective drugs. Antimicrobial peptides (AMPs) of the innate immune system are promising candidates for a role of such novel antibiotics. However, some cytotoxicity of AMPs toward host cells limits their active implementation in medicine and forces attempts to design numerous structural analogs of the peptides with optimized properties. An alternative route for the successful AMPs introduction may be their usage in combination with conventional antibiotics. Synergistic antibacterial effects have been reported for a number of such combinations, however, the molecular mechanisms of the synergy remain poorly understood and little is known whether AMPs cytotoxicy for the host cells increases upon their application with antibiotics. Our study is directed to examination of a combined action of natural AMPs with different structure and mode of action (porcine protegrin 1, caprine bactenecin ChBac3.4, human alpha- and beta-defensins (HNP-1, HNP-4, hBD-2, hBD-3), human cathelicidin LL-37), and egg white lysozyme with varied antibiotic agents (gentamicin, ofloxacin, oxacillin, rifampicin, polymyxin B, silver nanoparticles) toward selected bacteria, including drug-sensitive and drug-resistant strains, as well as toward some mammalian cells (human erythrocytes, PBMC, neutrophils, murine peritoneal macrophages and Ehrlich ascites carcinoma cells). Using "checkerboard titrations" for fractional inhibitory concentration indexes evaluation, it was found that synergy in antibacterial action mainly occurs between highly membrane-active AMPs (e.g., protegrin 1, hBD-3) and antibiotics with intracellular targets (e.g., gentamicin, rifampcin), suggesting bioavailability increase as the main model of such interaction. In some combinations modulation of dynamics of AMP-bacterial membrane interaction in presence of the antibiotic was also shown. Cytotoxic effects of the same combinations toward normal eukaryotic cells were rarely synergistic. The obtained data approve that combined application of antimicrobial peptides with antibiotics or other antimicrobials is a promising strategy for further development of new approach for combating antibiotic-resistant bacteria by usage of AMP-based therapeutics. Revealing the conventional antibiotics that increase the activity of human endogenous AMPs against particular pathogens is also important for cure strategies elaboration.