Organic chalcogen compounds have been investigated for several decades due to their often pronounced biological activities against parasites, microorganisms and cancer cells. Indeed, the field of natural Organic Sulfur Compounds (OSCs) has attracted tremendous attention, as Reactive Sulfur Species (RSS) such as allicin and polysulfanes from garlic or allylisocyanate (mustard oil) from various Brassica
plants exhibit interesting chemopreventive and possibly even therapeutic properties [1
]. Organoselenium compounds, in particular ebselen and its derivatives, pose a similar attraction in the Life Sciences, especially in the context of antioxidants [2
]. In contrast, organotellurium compounds have long remained marginalized in Biology, yet some of them, such as RT-01 and AS101, have also recently emerged from the chemical closet with some prominence [2
]. Not surprisingly, the field of biologically active organochalcogens has stimulated considerable efforts in chemical synthesis and has produced volumes of more or less exotic molecules [7
One of the predominant modes of action of many of these compounds is due to their ability to oxidize—rather selectively—the thiol groups of cysteine residues in pivotal cellular signalling proteins and enzymes, hence triggering processes which eventually may culminate in an antioxidant response, or the initiation of apoptosis or other forms of cell death. Intriguingly, the chalcogen-chalcogen bond often at the centre of such redox action is also found in the elemental forms of sulfur (S8
, common yellow form), selenium (Se8
, red allotrope) and tellurium (Te8
, dark grey), implying that such elemental modifications, from the perspective of reactivity, may themselves also be biologically active [9
]. Indeed, recent studies on inorganic polysulfides, such as the tetrasulfide S42−
, support this notion of simple, purely inorganic, “carbon free” yet highly active variants of chalcogens [10
]. Unfortunately, neither of the various elemental forms of any of the three chalcogens in question is soluble in water. Hence traditionally it has mostly been futile to apply elemental sulfur, selenium or tellurium in a biological context, with some notable exceptions, such as colloidal sulfur and its uses as fungicide in vineyards [13
Indeed, the apparent lack of solubility and hence bioavailability can be overcome with suspensions of small particles with diameters in the low or sub-micrometer range. We have recently shown that such particles can be obtained rather easily by physical or chemical methods and, in the case of selenium, even with the assistance of certain bacteria which readily generate natural, protein-coated selenium nanoparticles in adequate quality and yield. Most of these particles show some activity against microorganisms [15
]. Nanosized tellurium, in particular, appears to be rather active against organisms such as Escherichia coli
, Candida albicans
, Saccaromyces cerevisiae
and the model nematode Steinernema feltiae
]. While the nanosuspensions employed as part of such studies are rather promising as a first proof-of-concept for production and activity, they also have considerable drawbacks once used in practice. For instance, such liquid suspensions are difficult to maintain and to store for prolonged periods of time, they need to be kept under sterile conditions, are not particularly amenable to transport, and an adjustment of reliable concentrations is generally difficult. We have therefore undertaken a series of studies to produce easier-to-handle solid materials based on elemental chalcogens. Here we report our first results with the underlying NaLyre sequence of processing, which involves nanosizing, then lyophilizing and subsequently resuspending mannitol-stabilized chalcogen nanoparticles, and biological activities associated with them.
Overall, the results obtained as part of this feasibility study demonstrate that a sequence of nanosizing and lyophilization leads to ready-to-use materials which can be resuspended easily by short manual shaking to nanosuspensions with improved bioavailability. In this study, such a NaLyRe sequence results in nanosuspensions of sulfur, selenium and tellurium, which are generally of a good physical quality and show impressive activity against a range of microorganisms, notably C. albicans, E. coli and S. carnosus. The results obtained along this NaLyRe avenue and their implications will now be discussed in more detail.
From the perspective of sample preparation and quality, formation of the various chalcogen nanosuspensions through mechanical techniques such as HPH was generally straightforward. Still there were also some notable differences (Figure 1
). Sulfur was the least amenable of the three elements, its tendency to form cake-like materials and to sediment could be decreased by nanosizing, yet the average size of the sulfur particles could not be reduced much below 760 nm. Moving down the Periodic Table, the ability to nanosize seems to improve while applying the same experimental parameters. Under these conditions, selenium could be sized to an average particle diameter of 210 nm, while tellurium particles showed an average diameter of 170 nm. Similarly, the ability to lyophilize and to resuspend was most pronounced for the selenium and tellurium particles, while the resuspensions of the sulfur particles were stable, yet also rather “milky” (Figure 3
From a more practical and applied perspective, these findings are rather intriguing as they represent a strategy to convert solid elemental sulfur, selenium and tellurium into powders of a similar elemental composition, yet with good suspension properties and hence applicability and activity in biological systems. Once lyophilized, the chalcogen nanoparticles form easy-to-handle fluffy cakes (Figure 3
a) which require short manual resuspension times and maintain the size characteristics of the origin suspensions (Figure 3
and Figure 4
). Indeed, it appears that most particles retain their initial size and shape during the lyophilization/resuspension process, and that aggregation is not an encumbering issue. The respective ZPs, employed here as indicators of the stability of colloidal dispersions, also remain mostly unaffected by the lyophilization and resuspension procedure, and may even increase slightly. Unlike earlier liquid preparations, these lyophilized powders are considerably lighter, easier to store and to transport and do not need to be used up swiftly as they are dry and there is no danger of fouling. There is also no issue with leaks or spills which are common when handling liquid samples. Eventually, it is now feasible to prepare and store larger quantities of chalcogen nanoparticles of good quality and to resuspended them if, when and where desired for a range of possible applications, for instance in the fields of Medicine, Agriculture, Cosmetics and conceivably even in Nutrition, as may be applicable for selenium. Figure 11
provides a brief schematic illustration of the NaLyRe sequence and the potential medium-term applications associated with it.
Still, there are some issues which need to be addressed as part of subsequent studies. One of them is the use of stabilizers such as Plantacare®
to prevent aggregation in solution and mannitol as “cryoprotectant”. These components of the formulation are critical in providing protection against the stresses involved during the freeze-drying process. Indeed, without such stabilizers the formulation may be damaged in two ways. Firstly, particles at higher concentrations tend to agglomerate and to fuse. Secondly, the formation of ice crystals exerts mechanical stresses which destabilize the system [30
]. These effects in the absence of protective materials were, in fact, observed in this study as illustrated in Figure 2
. Here, the choice and concentration of protectant, as well as the size of particles affected are of importance. At concentrations of mannitol ranging from 1% to 5%, LD measurements show little impact of this apparent “cryoprotectant” on the agglomeration of larger particles. At higher concentrations of mannitol (e.g., at 20%), the integrity of the smaller size particles is maintained, and the agglomerations are also reduced. This can be explained by the properties of mannitol and similar cryoprotectants, which are able to form a protective matrix around the nanoparticles, isolating them as an unfrozen segment which in turn prevents the particles from agglomeration [31
Eventually, the stabilizers employed in this study are well established in the literature along with other sugars [20
]. Hence future studies may investigate the use of such agents in more detail and also consider alternatives. Trehalose, for instance, could possibly decrease the amount of cyroprotectant used, yet trehalose may complicate the composition and activity of the samples and is more expensive than common sugars, an economic aspect which may need to be considered as part of any practical application [20
]. Notably, certain sugars may also serve as nutrients, and this may be counterproductive in the context of antimicrobial activity. It is, therefore, worthwhile to investigate alternative stabilizers which may be either more effective, less problematic and perhaps also more readily available. It may even be possible to identify “two-in-one” agents able to substitute simultaneously for both stabilizers, Plantacare®
and mannitol, or to venture into agents which are “waste” or themselves biologically active for the additional “kick” [33
]. In any case, the NaLyRe sequence represents a major improvement in the production and handling of otherwise insoluble or sparingly soluble materials and, indeed, one may now consider possible medical and agricultural applications in earnest.
From a biological perspective, the initial attempts with mannitol have already enabled lyophilization and resuspension with considerable biological activities observed for the resuspended samples against the microbes tested. Among the chalcogens, tellurium appears to be most toxic overall. It was least effective against S. cerevisiae
, reducing the growth by just 20% at a formal concentration of 2000 µM (Figure 6
). For C. albicans
this activity was somewhat higher, with significant reductions already at a formal concentration of 1000 µM (Figure 7
). The chalcogens were particularly effective against the two bacteria investigated, especially for Gram-positive S. carnosus
where the growth was reduced by 50% in the presence of 2000 µM for selenium (Figure 8
and Figure 9
). In the case of the multicellular nematodes, tellurium was also active and, at a concentration of 200 µM, reduced growth to 60%. While the activity of selenium is often comparable to the one of tellurium, selenium was more active against the nematodes with a reduction of viability to below 50% of the control, while sulfur achieved a reduction to 70% (Figure 10
In Medicine, such resuspended particles therefore may be employed against topical infections, for instance in the case of skin, mucous, nails and the gastrointestinal tract. Here, the activity of the more active tellurium particles is of special interest. Many organotellurium compounds, as well as simple tellurium salts, often show considerable activity against pathogenic organisms, in some instances even re-sensitizing drug resistant strains against common antibiotics [34
]. Yet tellurium is also a fairly toxic element per se, and any application, even a topical one, clearly requires further investigations to exclude any unwanted side effects [35
]. The particular particulate structure may actually be a benefit rather than drawback in this context, as it almost rules out the kind of systemic uptake and distribution characteristic of soluble, toxic organotellurium compounds and, at the same time, may provide a slowly releasing system for certain reactive tellurium species (RTeS).
General toxicity is less of an issue with the trace element selenium. While the selenium nanoparticles may be oxidized or reduced as well to release diverse reactive selenium species (RSeS), selenides (H2
Se), selenite (SeO32−
) and selenate (SeO42−
) are readily detoxified and even utilized by the human body. The activity of the selenium-based nanosuspensions is therefore rather stimulating, especially in the context of C. albicans
, as yeasts are commonly known to be rather sensitive against this chalcogen and its diverse compounds. Here, the resuspended selenium particles, at a concentration of 2000 µM, reduce growth by 40%. Indeed, certain anti-dandruff shampoos contain selenium, and chemically speaking, the rather unusual mixed sulfur-selenium ring structures at the centre of such activity are not that different from the Se8
rings found in the selenium nanoparticles [37
]. Similarly, the resuspended selenium particles were also active against both strains of bacteria, at a formal concentration of 2000 µM reducing growth of S. carnosus
In any case, the precise mode(s) of action of these particles, against microbes and also in more complex organisms—from S. feltiae to humans—needs to be studied in considerable detail as part of future investigations and also to rule out any undesired or detrimental side effects of this material. Based on the literature available to date and on previous studies, it is feasible that such chalcogen particles act via a combination of mechanisms, which may involve more general physical interactions of the particles with cells and organelles, rather specific surface interactions—such as binding of and to proteins and enzymes, a specific surface chemistry of the chalcogens as well as a slow release of chalcogen-based molecules, such as the kind of inorganic polysulfides (Sx2−) mentioned in the Introduction.
From a wider perspective, and besides possible applications as antimicrobials in Medicine, resuspendable powders of selenium and sulfur may also be of interest in the field of Agriculture. As mentioned briefly in the Introduction, colloidal sulfur has a long tradition in the treatment of grapevines and a similar, perhaps more effective treatment could be envisaged for the nanosuspensions [13
]. Similarly, selenium may also be applied, obviously under considerably more controlled conditions and in considerably lower amounts. Here, sulfur, as well as selenium, may not only protect the plant from (microbial, nematode) predators, these particles may also enrich the soil, and, by slowly degrading, may serve as a reservoir of an inorganic fertilizer and eventually, in the case of selenium, even as trace element enrichment which may be beneficial along the nutritional chain.
In the medium term, the choice of particles and stabilizers will depend on the specific applications under consideration, and native suspensions—which initially are also sterile due to the manufacturing process—as well as resuspended lyophilized preparations seem to be quite stable and may be considered.
In summary, and without much suspense, we have been able to demonstrate that resuspension of ready-to-use lyophilized powders of sulfur, selenium and tellurium nanosuspensions on demand results in biologically active suspensions, albeit not solutions. The focus of this investigation has been on the proof-of-principle, and to evaluate the general feasibility of the underlying NaLyRe sequence.
Subsequent studies obviously need to investigate the various aspects of production, stability and wider applications of this approach and its products in considerably more detail. The choice of stabilizers and “cryo-protectants” such as Plantacare®
and mannitol, in particular, needs to be considered from the perspective of large-scale production, activity, side-effects, economy and environmental impact. Alternatives, for instance derived from agricultural waste or with specific, desirable biological activities may be of particular interest here [33
]. The mode(s) of action also need to be considered in considerably more detail, since these materials may impact on living organisms in various, physical, chemical, biochemical and physiological aspects. Such interactions, or nanotoxicity itself, may result in possible limitations due to adverse side effects. Nonetheless, as the examples of certain anti-dandruff formulations and colloidal sulfur illustrate, there is considerable potential stowed away in the elemental forms of these chalcogens, and unlocking this potential may be possible using nanotechnology.
Admittedly, these elemental forms may appear as rather “primitive” in a more biological context, especially when considering the numerous biologically active sulfur, selenium and tellurium compounds already known. Still, elemental forms have the major advantage that they do not carry the ballast of organic groups which may become modified, released or otherwise problematic. Indeed, the considerable recent interest in biologically active polysulfides, such as the tetrasulfide (S42−
), demonstrates that “chalcogen only” substances can play a significant role in biological systems [12
]. One may therefore speculate that S8
rings exposed on the surface of a mighty, at the same time slowly moving and degrading and chalcogen-releasing particle might exhibit considerable activity. Eventually, and with the NaLyRe sequence now available, other materials, such as sparingly or insoluble natural materials or products, parts of plants and even waste, may be processed in the same manner, therefore widening the scope of this approach and providing access to an even wider field of materials and applications [33