Elvira Rozhina

Halloysite Nanotubes: Controlled Access and Release by Smart Gates

Hollow halloysite nanotubes have been used as nanocontainers for loading and for the triggered release of calcium hydroxide for paper preservation. A strategy for placing end-stoppers into the tubular nanocontainer is proposed and the sustained release from the cavity is reported. The incorporation of Ca(OH)2 into the nanotube lumen, as demonstrated using transmission electron microscopy (TEM) imaging and Energy Dispersive X-ray (EDX) mapping, retards the carbonatation, delaying the reaction with CO2 gas. This effect can be further controlled by placing the end-stoppers. The obtained material is tested for paper deacidification. We prove that adding halloysite filled with Ca(OH)2 to paper can reduce the impact of acid exposure on both the mechanical performance and pH alteration. The end-stoppers have a double effect: they preserve the calcium hydroxide from carbonation, and they prevent from the formation of highly basic pH and trigger the response to acid exposure minimizing the pH drop-down. These features are promising for a composite nanoadditive in the smart protection of cellulose-based materials.

Cell surface engineering with polyelectrolyte-stabilized magnetic nanoparticles: A facile approach for fabrication of artificial multicellular tissue-mimicking clusters

Regenerative medicine requires new ways to assemble and manipulate cells for fabrication of tissue-like constructs. Here we report a novel approach for cell surface engineering of human cells using polymer-stabilized magnetic nanoparticles (MNPs). Cationic polyelectrolyte-coated MNPs are directly deposited onto cellular membranes, producing a mesoporous semi-permeable layer and rendering cells magnetically responsive. Deposition of MNPs can be completed within minutes, under cell-friendly conditions (room temperature and physiologic media). Microscopy (TEM, SEM, AFM, and enhanced dark-field imaging) revealed the intercalation of nanoparticles into the cellular microvilli network. A detailed viability investigation was performed and suggested that MNPs do not inhibit membrane integrity, enzymatic activity, adhesion, proliferation, or cytoskeleton formation, and do not induce apoptosis in either cancer or primary cells. Finally, magnetically functionalized cells were employed to fabricate viable layered planar (two-cell layers) cell sheets and 3D multicellular spheroids.

Nanohydrogel Formation within the Halloysite Lumen for Triggered and Sustained Release

An easy strategy to obtain nanohydrogels within the halloysite nanotube (HNTs) lumen was investigated. Inorganic reverse micelles based on HNTs and hexadecyltrimethylammonium bromides were dispersed in chloroform, and the hydrophilic cavity was used as a nanoreactor to confine the gel formation based on alginate cross-linked by calcium ions. Spectroscopy and electron microscopy experiments proved the confinement of the polymer into the HNT lumen and the formation of calcium-mediated networks. Biological tests proved the biocompatibility of the hybrid hydrogel. The nanogel in HNTs was suitable for drug loading and sustained release with the opportunity of triggered burst release by chemical stimuli. Here, we propose a new strategy based on inorganic reverse micelles for nanohydrogel formation, which are suitable for industrial and biological applications as well as for selective and triggered adsorption and/or release.

Targeting microbial biofilms using Ficin, a nonspecific plant protease

Biofilms, the communities of surface-attached bacteria embedded into extracellular matrix, are ubiquitous microbial consortia securing the effective resistance of constituent cells to environmental impacts and host immune responses. Biofilm-embedded bacteria are generally inaccessible for antimicrobials, therefore the disruption of biofilm matrix is the potent approach to eradicate microbial biofilms. We demonstrate here the destruction of Staphylococcus aureus and Staphylococcus epidermidis biofilms with Ficin, a nonspecific plant protease. The biofilm thickness decreased two-fold after 24 hours treatment with Ficin at 10 μg/ml and six-fold at 1000 μg/ml concentration. We confirmed the successful destruction of biofilm structures and the significant decrease of non-specific bacterial adhesion to the surfaces after Ficin treatment using confocal laser scanning and atomic force microscopy. Importantly, Ficin treatment enhanced the effects of antibiotics on biofilms-embedded cells via disruption of biofilm matrices. Pre-treatment with Ficin (1000 μg/ml) considerably reduced the concentrations of ciprofloxacin and bezalkonium chloride required to suppress the viable Staphylococci by 3 orders of magnitude. We also demonstrated that Ficin is not cytotoxic towards human breast adenocarcinoma cells (MCF7) and dog adipose derived stem cells. Overall, Ficin is a potent tool for staphylococcal biofilm treatment and fabrication of novel antimicrobial therapeutics for medical and veterinary applications.

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells

Many ribonucleases (RNases) are considered as promising tools for antitumor therapy because of their selective cytotoxicity toward cancer cells. Binase, the RNase from Bacillus pumilus, triggers apoptotic response in cancer cells expressing RAS oncogene which is mutated in a large percentage of prevalent and deadly malignancies including colorectal cancer. The specific antitumor effect of binase toward RAS-transformed cells is due to its direct binding of RAS protein and inhibition of downstream signaling. However, the delivery of proteins to the intestine is complicated by their degradation in the digestive tract and subsequent loss of therapeutic activity. Therefore, the search of new systems for effective delivery of therapeutic proteins is an actual task. This study is aimed to the investigation of antitumor effect of binase immobilized on natural halloysite nanotubes (HNTs). Here, we have developed the method of binase immobilization on HNTs and optimized the conditions for the enzyme loading and release (i); we have found the non-toxic concentration of pure HNTs which allows to distinguish HNTs- and binase-induced cytotoxic effects (ii); using dark-field and fluorescent microscopy we have proved the absorption of binase-loaded HNTs on the cell surface (iii) and demonstrated that binase-halloysite nanoformulations possessed twice enhanced cytotoxicity toward tumor colon cells as compared to the cytotoxicity of binase itself (iv). The enhanced antitumor activity of biocompatible binase-HNTs complex confirms the advisability of its future development for clinical practice.

Antimicrobial Effects of Sulfonyl Derivative of 2(5H)-Furanone against Planktonic and Biofilm Associated Methicillin-Resistant and -Susceptible Staphylococcus aureus

The gram-positive opportunistic bacterium Staphylococcus aureus is one of the most common causatives of a variety of diseases including skin and skin structure infection or nosocomial catheter-associated infections. The biofilm formation that is an important virulence factor of this microorganism renders the antibiotic therapy ineffective, because biofilm-embedded bacteria exhibit strongly increased tolerance to antimicrobials. Here, we describe a novel 3-chloro-5(S)-[(1R,2S,5R)-2-isopropyl-5-methylcyclohexyloxy]-4-[4-methylphenylsulfonyl]-2(5H)-furanone (F105), possessing a sulfonyl group and l-menthol moiety. Minimal inhibitory and bactericidal concentration values (MIC and MBC) of F105 were 10 and 40 mg/L, respectively, suggesting F105 biocidal properties. F105 exhibits pronounced activity against biofilm-embedded S. aureus and increases the efficacy of aminoglycosides (amikacin, gentamicin, and kanamycin) and benzalkonium chloride with fractional inhibitory concentration index values of 0.33–0.44 and 0.29, respectively, suggesting an alternative external treatment option, e.g., for wound infections. Moreover, low concentrations (0.5–1.3 mg/L) of F105 reduced the MICs of these antimicrobials twofold. By using confocal laser scanning microscopy and CFU counting, we show explicitly that F105 also restores the antimicrobial activity of gentamicin and ampicillin against S. aureus biofilms by several orders of magnitude. Biofilm structures were not destroyed but sterilized, with embedded cells being almost completely killed at twofold MBC. While F105 is quite toxic (CC50/MBC ratio 0.2), our data suggest that the F105 chemotype might be a promising starting point for the development of complex topical agents for combined anti-staphylococcal biofilm-therapies restoring the efficacy of some antibiotics against difficult to treat S. aureus biofilm.

Fluorescence and Cytotoxicity of Cadmium Sulfide Quantum Dots Stabilized on Clay Nanotubes

Quantum dots (QD) are widely used for cellular labeling due to enhanced brightness, resistance to photobleaching, and multicolor light emissions. CdS and CdxZn1−xS nanoparticles with sizes of 6–8 nm were synthesized via a ligand assisted technique inside and outside of 50 nm diameter halloysite clay nanotubes (QD were immobilized on the tube’s surface). The halloysite–QD composites were tested by labeling human skin fibroblasts and prostate cancer cells. In human cell cultures, halloysite–QD systems were internalized by living cells, and demonstrated intense and stable fluorescence combined with pronounced nanotube light scattering. The best signal stability was observed for QD that were synthesized externally on the amino-grafted halloysite. The best cell viability was observed for CdxZn1−xS QD immobilized onto the azine-grafted halloysite. The possibility to use QD clay nanotube core-shell nanoarchitectures for the intracellular labeling was demonstrated. A pronounced scattering and fluorescence by halloysite–QD systems allows for their promising usage as markers for biomedical applications.

Complex interbacterial interactions in mixed biofilms as a key determinant of their antimicrobial treatment efficacy

Biofilms are formed by closely adjacent microorganisms embedded into an extracellular matrix this way providing them with strong protection from antimicrobials, which is often further reinforced in polymicrobial biofilms. Despite of the well-known antagonistic interactions between S. aureus and P. aeruginosa, the most common pathogens causing various nosocomial infections, they often form mixed consortia characterized by increased pathogenicity and delayed recovery in comparison with single species infections. Here we show that, while S. aureus could successfully avoid a number of antimicrobials by embedding into the biofilm matrix of P. aeruginosa despite of their antagonism, the very same consortium was characterized by 10-fold higher susceptibility to broad-spectrum antimicrobials compared to monocultures. Moreover, quantitatively similar increase in antimicrobials susceptibility could be achieved when P. aeruginosa was introduced into S. aureus biofilm, compared to S. aureus monoculture. In a reverse experiment, intervention of S. aureus into the mature P. aeruginosa biofilm significantly increased the efficacy of ciprofloxacin against P. aeruginosa. A broader perspective is provided by antagonistic bacteria intervention into already preformed monoculture biofilms leading to the considerable enhancement of their antibiotic susceptibility. We believe that this approach has a strong potential of further development towards innovative treatment of biofilm-associated infections such as transplantation of the skin residential microflora to the wounds and ulcers infected with nosocomial pathogens to speed up their microbial decontamination.While in biofilms bacteria are embedded into an extracellular matrix which forms inaccessible barrier for antimicrobials thereby drastically increasing the concentrations of antibiotics required for treatment. Here we show that the susceptibility of S. aureus and P. aeruginosa to antibiotics in mixed biofilms significantly differs from monoculture biofilms depending on both conditions and chosen antimicrobial agents. While S. aureus could completely avoid vancomycin, ampicillin and ceftriaxone by embedding into the biofilm of P. aeruginosa, the very same consortium was characterized by 10–fold increase in susceptibility to broad-spectrum antimicrobials like ciprofloxacin and aminoglycosides compared to monocultures. These data clearly indicate that efficient treatment of biofilm-associated mixed infections requires antimicrobials active against both pathogens, since the interbacterial antagonism would enhance the efficacy of treatment. Moreover, similar increase in antibiotics efficacy was observed when P. aeruginosa suspension was added to the mature S. aureus biofilm, compared to S. aureus monoculture, and vice versa. These findings open promising perspectives to increase the antimicrobial treatment efficacy of the wounds infected with nosocomial pathogens by the transplantation of the skin residential microflora.