Calcium phosphate nanoparticles as intrinsic inorganic antimicrobials: mechanism of action.

This is the final report of the study aimed at assessing the antimicrobial activity of calcium phosphate (CP) nanoparticles delivered in the form of hydroxyapatite (HAp) or amorphous CP (ACP) and understanding the fundamental principles behind their mechanisms of action. Not responding to propidium iodide and causing no gross morphological changes except moderate stress-induced filamentation in Escherichia coli (E. coli), CP nanoparticles were shown to be bacteriostatic, not bactericidal. Also, the lack of expression of genes involved in DNA repair indicated no genotoxic activity. In contrast, the softening of amide infrared bands and the partial dissociation of lipopolysaccharide structures comprising the membrane of Gram-negative Pseudomonas aeruginosa (P. aeruginosa) was detected in a vibrational spectroscopic analysis of the nanoparticle/bacterium interaction. Similarly, the inhibition of the growth of Staphylococcus aureus (S. aureus) was paralleled by a reduced integrated intensity and the softening of the C = O ester carbonyl stretch in lipoteichoic acid, a major component of the Gram-positive cell membrane. Electron microscopy analyses confirmed that changes to the cell membrane are a major mode of action of CP nanoparticles. While HAp got internalized by E. coli significantly more than ACP, the membrane damage was more pronounced in ACP-treated bacteria, which was explained by the higher surface reactivity of ACP. HAp nanoparticles decreased the activity of overexpressed efflux pumps in methicillin-resistant S. aureus, suggesting that they may hijack these pumps and use them to enter the cell without producing any visible damage to the membrane, thus acting on the cell from the inside out, as opposed to ACP, whose action is mostly external in mechanism. This may explain why HAp, unlike ACP, suppresses the mechanisms of resistance in methicillin- and multidrug-resistant S. aureus and P. aeruginosa, respectively. The findings of this study will be essential in the optimization of these nanoparticles for becoming an alternative to less biocompatible inorganics and small molecule antibiotics in the global effort to curb the rising resistance of bacterial pathogens to the existing therapies.

Aflatoxin and ochratoxin production by Aspergillus species under ex vivo conditions.

Aspergillus species are increasingly important human pathogens. It is not known whether toxic metabolites of many of these pathogenic species can act as virulence factors in aspergillosis. We examined isolates of aflatoxin and ochratoxin-producing species for toxin production in ex vivo conditions. Seven of the 21 aflatoxin-producing isolates screened produced aflatoxin at 35 and 37 degrees C on the general medium yeast extract sucrose agar (YES). However, none of them produced toxin at these temperatures on brain heart infusion agar (BHA), a medium that mimics human tissue, or on BHA with modified pH or sugar levels. Six of the 12 ochratoxin-producing isolates examined produced toxin at 35 degrees C on YES. All three isolates of A. alliaceus produced ochratoxin on BHA or modified BHA at 37 degrees C. One strain of A. pseudoelegans produced a minute amount of ochratoxin on modified BHA at 37 degrees C. These data indicate that aflatoxin is an unlikely virulence, factor but that ochratoxin may be a potential virulence factor in aspergillosis.

Targeted magnetic separation of biomolecules and cells using earthicle-based ferrofluids.

Targeting specific molecular or cell populations within single tissues or multicomponent in vitro systems is a most sought goal in biomedicine. Here we report on targeted magnetic separation of cells and biomolecules using a ferrofluid comprising superparamagnetic iron-oxide/silicate/carbon core/shell/crust nanoparticles in combination with a handheld, 2.5 cm3 NdFeB magnet (≤180 mT) and one minute exposure time. Ferrofluids were highly effective at separating (i) biomolecules, (ii) bacteria and (iii) eukaryotic cells from solutions, and they also exhibited selectivity in the separation of all three families of entities. Specifically, they were more effective at separating the negatively charged protein, albumin in the presence of the external magnetic field, but were more effective at precipitating the positively charged protein, lysozyme without the application of the external field. Because of the more effective sorption of proteins than carbohydrates on carbon and the shielding of peptidoglycans by the transmembrane proteins and hydrophilic heads of the outer membrane amphiphiles in Gram-negative bacteria, they were separated more effectively than their Gram-positive counterparts. Ferrofluids were also more efficient at separating the clinical isolate, methicillin-resistant version of S. aureus (MRSA) than its regular, lab strain and the effect is thought to be due to structural changes to the cell envelope caused by the overexpression of efflux pumps or by the higher rate of conjugation conditioning horizontal gene transfer in MRSA than in the regular, nonresistant strain. Ferrofluids also displayed a greater affinity for the cancer cells than for the normal, primary cells and allowed for targeted separation of the former after the cells were allowed to uptake the nanoparticles for 24 h. This selectivity should allow for an effective separation of cancer cells interspersed within a healthy cell population. Interaction with bacterial and eukaryotic cells was driven neither by electrostatic attraction nor chemisorption, but by weaker, van der Waals and π-interactions. Adsorption was also endothermic, irreversible for the most part, and more favorable at high concentrations, as inferred by comparison with Langmuir, Freundlich, Temkin and Dubinin-Radushkevich isotherms. These targeted effects are relevant for numerous fields of biomedicine and biotechnologies and require further insight for optimization and translation.

Brain and bone cancer targeting by a ferrofluid composed of superparamagnetic iron-oxide/silica/carbon nanoparticles (earthicles).

Despite the advances in molecularly targeted therapies, delivery across the blood-brain barrier (BBB) and the targeting of brain tumors remains a challenge. Like brain, bone is a common site of metastasis and requires therapies capable of discerning the tumor from its healthy cellular milieu. To tackle these challenges, we made a variation on the previously proposed concept of the earthicle and fabricated an aqueous, surfactant-free ferrofluid containing superparamagnetic iron oxide nanoparticles (SPIONs) coated with silicate mesolayers and carbon shells, having 13 nm in size on average. Nanoparticles were synthesized hydrothermally and characterized using a range of spectroscopic, diffractometric, hydrodynamic and electron microscopy techniques. The double coating on SPIONs affected a number of physicochemical and biological properties, including colloidal stability and cancer targeting efficacy. Nanoparticles decreased the viability of glioblastoma and osteosarcoma cells and tumors more than that of their primary and non-transformed analogues. They showed a greater preference for cancer cells because of a higher rate of uptake by these cells and a pronounced adherence to cancer cell membrane. Even in an ultralow alternate magnetic field, nanoparticles generated sufficient heat to cause tumor death. Nanoparticles in MDCK-MDR1 BBB model caused mislocalization of claudin-1 at the tight junctions, underexpression of ZO-1 and no effect on occludin-1 and transepithelial resistance. Nanoparticles were detected in the basolateral compartments and examination of LAMP1 demonstrated that nanoparticles escaped the lysosome, traversed the BBB transcellularly and localized to the optic lobes of the third instar larval brains of Drosophila melanogaster. The passage was noninvasive and caused no adverse systemic effects to the animals. In conclusion, these nanoparticulate ferrofluids preferentially bind to cancer cells and, hence, exhibit a greater toxicity in these cells compared to the primary cells. They are also effective against solid tumors in vitro, can cross the BBB in Drosophila, and are nontoxic based on the developmental studies of flies raised in ferrofluid-infused media. STATEMENT OF SIGNIFICANCE: We demonstrate that a novel, hydrothermally synthesized composite nanoparticle-based ferrofluid is effective in reducing the viability of osteosarcoma and glioblastoma cells in vitro, while having minimal effects on primary cell lines. In 3D tumor spheroids, nanoparticles greatly reduced the metastatic migration of cancer cells, while the tumor viability was reduced compared to the control group by applying magnetic hyperthermia to nanoparticle-treated spheroids. Both in vitro and in vivo models of the blood-brain barrier evidence the ability of nanoparticles to cross the barrier and localize to the brain tissue. These composite nanoparticles show great promise as an anticancer biomaterial for the treatment of different types of cancer and may serve as an alternative or addendum to traditional chemotherapies.

Colloids or powders: Which nanoparticle formulations do cells like more?

Understanding the difference in physicochemical properties and biological response between colloidal and powder formulations of identical materials is important before the given materials are used in a medical milieu. In this study we compared a set of biological effects of colloidal and powder formulations of composite nanoparticles comprising superparamagnetic iron oxide cores and silicate/carbon shells. Magnetic dipole interaction between adjacent nanoparticles was more pronounced in their powders than in their colloidal formulations. Nanoparticles delivered as powders were thus more responsive to the magnetic field, but exhibited reduced uptake in bone and brain cancer cells, including K7M2 osteosarcoma line and U87 and E297 glioblastoma lines. Specifically, while the alternate magnetic field elicited a more rapid heat generation in cell culture media supplemented with the magnetic powders, the nanoparticles dispersed in the same media were uptaken by the cancer cells more copiously. The cellular uptake proved to be more crucial in defining the effect on cell survival, given that suspended formulations elicited a greater degree of cancer cell death in the magnetic field compared to the powder-containing formulations. Because of this effect, colloidal formulations were able to target cancer cells more effectively than the powders: they reduced the viability of all three tested cancer cell lines to a significantly greater degree that the viability of the normal, MDCK-MDR1 cell line. It is concluded that better uptake profile can make up for the lower heating rate in the AC field and lead to a more effective magnetic hyperthermia therapy. These results also demonstrate that the direct delivery of ferrofluids is more optimal than the administration of their constitutive particles as powders.

Calcium phosphate nanoparticles as intrinsic inorganic antimicrobials: In search of the key particle property.

One of the main goals of materials science in the 21st century is the development of materials with rationally designed properties as substitutes for traditional pharmacotherapies. At the same time, there is a lack of understanding of the exact material properties that induce therapeutic effects in biological systems, which limits their rational optimization for the related medical applications. This study sets the foundation for a general approach for elucidating nanoparticle properties as determinants of antibacterial activity, with a particular focus on calcium phosphate nanoparticles. To that end, nine physicochemical effects were studied and a number of them were refuted, thus putting an end to frequently erred hypotheses in the literature. Rather than having one key particle property responsible for eliciting the antibacterial effect, a complex synergy of factors is shown to be at work, including (a) nanoscopic size; (b) elevated intracellular free calcium levels due to nanoparticle solubility; (c) diffusivity and favorable electrostatic properties of the nanoparticle surface, primarily low net charge and high charge density; and (d) the dynamics of perpetual exchange of ultrafine clusters across the particle/solution interface. On the positive side, this multifaceted mechanism is less prone to induce bacterial resistance to the therapy and can be a gateway to the sphere of personalized medicine. On a more problematic side, it implies a less intense effect compared to single-target molecular therapies and a difficulty of elucidating the exact mechanisms of action, while also making the rational design of theirs for this type of medical application a challenge.

Molecular Recognition of M1-Linked Ubiquitin Chains by Native and Phosphorylated UBAN Domains.

Although the Ub-binding domain in ABIN proteins and NEMO (UBAN) is highly conserved, UBAN-containing proteins exhibit different Ub-binding properties, resulting in their diverse biological roles. Post-translational modifications further control UBAN domain specificity for poly-Ub chains. However, precisely, how the UBAN domain structurally confers such functional diversity remains poorly understood. Here we report crystal structures of ABIN-1 alone and in complex with one or two M1-linked di-Ub chains. ABIN-1 UBAN forms a homo-dimer that provides two symmetrical Ub-binding sites on either side of the coiled-coil structure. Moreover, crystal structures of ABIN1 UBAN in complex with di-Ub chains reveal a concentration-dependency of UBAN/di-Ub binding stoichiometry. Analysis of UBAN/M1-linked di-Ub binding characteristics indicates that phosphorylated S473 in OPTN and its corresponding phospho-mimetic residue in ABIN-1 (E484) are essential for high affinity interactions with M1-linked Ub chains. Also, a phospho-mimetic mutation of A303 in NEMO, corresponding to S473 of OPTN, increases binding affinity for M1-linked Ub chains. These findings are in line with the diverse physiological roles of UBAN domains, as phosphorylation of OPTN UBAN is required to enhance its binding to Ub during mitophagy.

On Grounds of the Memory Effect in Amorphous and Crystalline Apatite: Kinetics of Crystallization and Biological Response.

Memory effects, despite being intrinsic to biological systems, are rarely potentiated in biomaterials. By exploring the transition between amorphous calcium phosphate (ACP) and hydroxyapatite (HAp) from different empirical angles, here, we attempt to set the basis for elicitation of structural memory effects in CPs. Two CPs precipitated under different degrees of saturation (DS), yielding HAp at a low DS and ACP at a high DS, were shown to evolve into structures with a high level of crystallographic similarity after their prolonged aging in the solution and served as the basis for this study. Amorphous-to-crystalline transition was abrupt in both precipitates, indicating an autocatalytic process preceded by considerable nucleation lag times, but it was more dynamic and proceeded in multiple stages in the precipitate formed at a higher DS, involving a greater degree of lattice rearrangements. ACP was found to exist in one of the two stoichiometrically and crystallographically different forms, one of which, amounting to ≥60 wt %, resembled tricalcium phosphate and transformed to HAp through the surface dissolution/reprecipitation mechanism and the other one, amounting to ≤20 wt %, was apatitic, enabling the transformation of ACP to HAp via martensitic, bulk lattice reordering phenomena. Large density of stacking faults was responsible for the comparatively high lattice strain, the property to which biogenic apatite owes its ability to accommodate foreign ions and act as a mineral reservoir for the body. Being the precursor for biogenic apatite during biomineralization and a thermodynamically logical intermediate in the ripening of HAp per the Ostwald law of stages, ACP proved to be more prone to structural transformation than the final and the most stable of the CP phases in this sequence of events: HAp. Amorphized upon gelation, two CPs transformed into HAp, albeit at different rates, which were higher for the material that had been crystalline prior to amorphization than for the one that had initially been amorphous, indicating the presence of a definite memory effect. The two HAp powders with different histories of formation also elicited different biological responses, including a Runx2 transcription factor expression in MC3T3-E1 osteoblasts, cell uptake efficiency, and antibacterial activity, extending the memory effect in HAp to the biological domain. The biological response was typically indistinct between the final products and their respective precursors but markedly different between the two products obtained by following different formation paths, confirming the presence of the given memory effect. It is suggested that the key to explaining the difference in the response between the materials differing in their route of formation lies in the direct dependence between the DS at which precipitation occurs and the rate of exchange of hydrated ions and ionic clusters across the particle surface in contact with a solution.

Calcium Phosphate Nanoparticles as Intrinsic Inorganic Antimicrobials: The Antibacterial Effect.

Cheap and simple to make, calcium phosphate (CP), thanks to its unusual functional pleiotropy, belongs to the new wave of abundant and naturally accessible nanomaterials applicable as a means to various technological ends. It is used in a number of industries, including the biomedical, but its intrinsic antibacterial activity in the nanoparticle form has not been sufficiently explored to date. In this study, we report on this intrinsic antibacterial effect exhibited by two distinct CP phases: an amorphous CP (ACP) and hydroxyapatite (HAp). The effect is prominent against a number of regular bacterial species, including Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa, but also their multidrug-resistant (MDR) analogues. Although ACP and HAp displayed similar levels of activity against Gram-negative organisms, ACP proved to be more effective against the Gram-positive ones, with respect to which HAp was mostly inert, yet this trend became reversed for the MDR strains. In addition to the intrinsic antimicrobial effect of CP nanoparticles, we have also observed a synergistic effect between the nanoparticles and certain antibiotics. Both forms of CP were engaged in a synergistic relationship with a variety of concomitantly delivered antibiotics, including ampicillin, kanamycin, oxacillin, vancomycin, minocycline, erythromycin, linezolid, and clindamycin, and enabled even antibiotics completely ineffective against particular bacterial strains to significantly suppress their growth. This relationship was complex; depending on a particular CP phase, bacterial strain and antibiotic, the antibacterial activity (i) intensified proportionally to the nanoparticle concentration, (ii) plateaued immediately after the introduction of nanoparticles in minute amounts, or (iii) exhibited concentration-dependent minima due to stress-induced biofilm formation. These findings present grounds for the further optimization of CP properties and maximization of this intriguing effect, which could in the long run make this material comparable in activity to the inorganics of choice for this application, including silver, copper, or zinc oxide, while retaining its superb safety profile and positive eukaryotic versus prokaryotic cell selectivity.

Disruption of NSD1 in Head and Neck Cancer Promotes Favorable Chemotherapeutic Responses Linked to Hypomethylation.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) represents a distinct classification of cancer with worse expected outcomes. Of the 11 genes recurrently mutated in HNSCC, we identify a singular and substantial survival advantage for mutations in the gene encoding Nuclear Set Domain Containing Protein 1 (NSD1), a histone methyltransferase altered in approximately 10% of patients. This effect, a 55% decrease in risk of death in NSD1-mutated versus non-mutated patients, can be validated in an independent cohort. NSD1 alterations are strongly associated with widespread genome hypomethylation in the same tumors, to a degree not observed for any other mutated gene. To address whether NSD1 plays a causal role in these associations, we use CRISPR-Cas9 to disrupt NSD1 in HNSCC cell lines and find that this leads to substantial CpG hypomethylation and sensitivity to cisplatin, a standard chemotherapy in head and neck cancer, with a 40% to 50% decrease in the IC50 value. Such results are reinforced by a survey of 1,001 cancer cell lines, in which loss-of-function NSD1 mutations have an average 23% decrease in cisplatin IC50 value compared with cell lines with wild-type NSD1Significance: This study identifies a favorable subtype of HPV-negative HNSCC linked to NSD1 mutation, hypomethylation, and cisplatin sensitivity. Mol Cancer Ther; 17(7); 1585-94. ©2018 AACR.