**2. Application of insecticides nanoformulations**

#### **2.1. Nanoemulsions**

merous losses that are caused by insects on agriculture, several chemicals have been used to kill them or inhibit their reproduction and feeding habits. Those classes of compounds are collectivity known as insecticides. These molecules are able to interfere in the insect metabo‐ lism. They alter is in such a way that the plague cannot feeds on the crop or the harvest or even reproduce anymore. The use of insecticides is described since ancient times, with docu‐ ments providing evidences as far as in the 16th century BC. The *Ebers Papyrus*, wrote by the Egyptians, reports several chemical and organic substances used against overcome fleas, gnats and biting flies among others [4]. Nowadays, the insecticides are widely employed around the world. Several known substances are extremely effective in controlling or even wiping out almost all important agricultural plagues. This multi-billion-dollar has an esti‐ mated production of 2 million metric tons of hundreds of chemical and biological different

Insecticides are used in different ways, based on the physical-chemical characteristics of the each chemical substance, the area that needs to be covered and the target. Typical applica‐ tion of insecticides in crops is made by spraying a solution, emulsion or colloidal suspension containing the active chemical compound, which is made by a vehicle which may be a hand pump, a tractor or even a plane. This mixture is prepared using a liquid as a carrier, usually water, to ensure a homogenous distribution. Other methods for applying insecticides are through foggers or granule baits embedded with the active compound, among others that are less used. However, due to several degradation processes, such as leaching or destruc‐ tion by light, temperature, microorganism or even water (hydrolysis), only a small amount of these chemical products reaches the target site. In this case, the applied concentrations of these compounds have been much higher than the required. On the other hand, the concen‐ tration that reaches its target might be lower than the minimum effective one. In general, de‐ pending of the weather and method of application, the amount of applied agrochemicals, as much as 90%, may not reach the target and so do not produce the desired biological re‐ sponse. For this reason, repeated application of pesticides become hence necessary to effi‐ cient control of target plagues, which increase the cost and might cause undesirable and serious consequences to the ecosystems, affecting human health [6]. Due to the lack of selec‐ tivity, their unrestrained use can also lead to the elimination of the natural enemies, what implies in the fast growth of plague population. Moreover, it often makes the insects resist‐

Another important point that needs attention is the formulation for the application of the in‐ secticide on the crops. There are several different classes of compounds, which sometimes do not match with a simple dilution in water and must be prepared by other means such as powders, emulsions or suspensions. Some kinds of formulations must be handled with more precaution, since it can severely contaminate workers on the field with small airborne

The advances in science and technology in the last decades were made in several areas of insecticide usage. It includes either the development of more effective and non-persistent pesticides and new ways of application, which includes controlled release formulations (CRFs). The endeavors are direct towards the successful application of those compounds on

products, with a budget of a US\$35 billion dollars worldwide [5].

522 Insecticides - Development of Safer and More Effective Technologies

ant to the pesticides.

solid particles that can be inhaled [7].

Casanova *et al*. [9] evaluated the production of a nicotine carboxylate nanoemulsion using a series of fatty acids (C10 – C18) and surfactant. The oil-in-water nanoemulsion showed a monomodal distribution of size, with mean particle sizes of 100nm. The bioactivity of the insecticide formulations was evaluated against adults of *Drosophila melanogaster* by assessing the lethal time 50 (LT50). They observed that the encapsulation efficiency decreased with in‐ creasing size of the fatty acids tested. The bioactivity followed the same trend, with better bioactivity when the chain length decreased. This would be readily attributed to the higher amount of active compound inside the nanoemulsion. For the smallest fatty acid emulsion used, the capric acid (C10) one, the greatest encapsulation efficiency was observed, but it had the lowest bioactivity. The results were explained in terms of lesser bioavailability of the insecticide in its active form due to increased stability of the organic salt formed between the insecticide and the fatty acid. This experiment highlights the necessity of developing differ‐ ent kinds of possible assembles between the active compounds and matrix, and extensively studying the interactions in nanoscale formulations, where sometimes nontrivial effects might be unexpectedly observed.

Wang *et al.*[10] developed an assemble of oil-in-water nanoemulsion (O/W) with 30 nm droplets by careful control of experiment conditions, using the neutral surfactant poly(oxy‐ ethylene) lauryl ether and methyl decanoate to encapsulate highly insoluble β-cypermeth‐ rin. The dissolution of the insecticide was enhanced. The stability tests were performed by spraying nanoemulsion in a glass slide and observing under polarizing light microscopy. They showed no apparent precipitate in nanoemulsions samples. These results were differ‐ ent from the ones obtained using a commercial β-cypermethrin formulation, with apparent signs of solid residues after 24 hours. This enhanced stability may be used to decrease the concentration of insecticides in commercial spray applications, without losing efficiency.

#### **2.2. Classical micro and nanoparticles**

Allan *et al.* [11] published the first report on a controlled release system of an insecticide through a polymeric encapsulation. Even so, at first the encapsulated systems were not so effective. Problems associated with controlled release and particle stability hindered their practical field application for some decades. In one of the first successful works in the field of pesticides encapsulation, Greene *et al.* [12] used poly (n-alkyl acrylates) (Intelimer®) to produce temperature-sensitive microcapsules of the organophosphate insecticide diazinon.. The active chemical was controlled release by increasing the ambient temperature above 30ºC, which is the melting temperature of the polymer,. Experiments were performed with Banded cucumber beetle *Diabrotica balteata* and Western corn rootworm *Diabrotica virgifera* as target insects at 20ºC and 32ºC, under and above the polymer melting point respectively. Mortality was compared to commercial granular formulation. At lower temperatures, the commercial formulation showed the best mortality. At higher temperatures the activity of the encapsulated formulation was better, showing about 90% of mortality for over 8 weeks. The commercial formulation had indeed lost some of its activity, presumably due to heat degradation.

clusion of poly(ε-caprolactone) (PCL) into the matrix in 50-80% weight were analyzed, with formation of microspheres of PLA/PCL blend with 20-120µm of the diameter, showing up to

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525

In contrast to conventional desire to produce compounds with extended residual activity, quick-release microcapsules are demanded in certain areas of agriculture. However, some‐ times it is also necessary a quick liberation of the active compound from the matrix after the application. The strong backbone might pose as a problem to effectively deliver. Studies per‐ formed by Tsuda *et al.* [18,19] have shown that is possible to assemble "self-bursting" micro‐ capsules that retain its form in water suspension, but easily burst after solvent evaporation. They used the interfacial polymerization method to assemble spherical polyurethane micro‐ capsules containing the insecticide pyriprofixen, obtaining particles with mean diameter of 23µm. The entrapment ratio was 99% for all formulations tested, greatly improving the solu‐ bility of the pesticide in water. According to the results, there is a correlation between the wall thickness of the microcapsules and the self-bursting phenomenon. Tuning this property

The effectiveness of encapsulated formulations, it is not restricted to extend the residual activi‐ ty of insecticides, but should also include the overcoming of problems associated with accu‐ mulation of recalcitrant organic pollutants that remains in ecosystems in amounts above the Maximum Residual Level (MRL). Therefore, it can be harmful to the environment and to peo‐ ple who might consume the treated crops. For instance, Guan *et al.* [20] encapsulated imidaclo‐ prid, a chloro-nicotinyl systemic and broad spectrum insecticide in a mixed sodium alginate/ chitosan microparticle through self-assembly layer-by-layer (LbL) methodology. The capsules showed a mean diameter of 7µm. Particles were impregnated with a photocatalyst made of SDS/TiO2/Ag, and the photocatalytic property and the insecticidal activity of the microcap‐ sule was evaluated. Prolonged residual activity of the encapsulated formulation was ob‐ served. The toxicity was higher in the *Martianus dermestoides* adult stage compared to the one of pure insecticide. In a field test with soybean [21], the nano-imidacloprid formulation prevent‐ ed the accumulation of the pesticide on the soybean leaves and soil. The results showed pro‐ nounced degradation over 25 days of trials when compared to commercial concentrate formulations, even though the initial concentration of both formulations was equivalent. In this way, regardless the initial effectiveness of the insecticide, safer levels of agrochemicals can

Besides the chemical compounds, the micro- and nanotechnology have also been developed and applied to microorganisms that need special protection or to improve their solubility in aqueous phase. For instance, Ramírez-Lepe *et al.* [22] developed an aluminium-carboxyme‐ thylcellulose microcapsule with photoprotective agents for holding a *Bacillus thuringiensis* serovar *israelensis* (B.t.i.) spore-toxin complex named δ-endotoxin. The protein produced by this gram-positive bacterium during sporulation is extremely toxic to larval stage of some mosquitoes and flies which are vectors for important tropical diseases such as malaria and dengue. The encapsulated formulation was tested for its UV irradiation protective efficiency

be obtained in less time, improving the safety of insecticide application.

**2.3. Entomopathogenic microorganisms encapsulated**

88,5% of insecticide release in aqueous media over a 48h period.

a controlled released can be achieved.

Latheef *et al.* [13] tested several different polymers such as poly (methyl methacrylate) (PMMA), ethyl cellulose, poly(α-methylstyrene) and cellulose acetate butyrate to produce microcapsules of the insecticide sulprofos. Ethyl cellulose formulations were the only ones that had shown good results against eggs and larvae of the tobacco budworm *Heliothis vires‐ cens* in cotton plants. The results were comparable to the ones obtained with the use of an emulsifable-concentrate (EC) commercial formulation of sulprofos.

In other to develop commercial formulation containing microencapsulated cyfluthrin, Ar‐ thur[14] evaluated its use against the rice weevil *Sitophilus oryzae* in stored wheat, for a peri‐ od of 8 months. Survival of beetles was statistically correlated with the concentration of the pyrethroid insecticide in the formulation. The average survival rate was only 12% when 4ppm was used, with constant activity throughout the entire experiment. This evidenced the controlled release of the substance over a long period of time.

In the work carried out by Quaglia *et al.* [15], a hydrophobic waxy prepared through a mix‐ ture of di- and triglycerides of PEG esters was used to construct microspheres containing the insecticide carbaryl. Microparticles was obtained with particle size ranging from 16 to 20µm. Controllable release dynamics depended on the amount of gelucire used, Studies of release profiles from the encapsulated formulation showed a lower vertical mobility of the insecti‐ cide when compared to a commercial nonencapsulated formation. This suggested that the controlled release profile of the microcapsules may be useful to avoid or minimize ground‐ water contamination.

Cao *et al.* [16] produced diffusion-controlled microcapsules with diameter ranging from 2 to 20µm with encapsulated acetamiprid, an alkaline and high temperature-sensitive insecti‐ cide, using tapioca starch as matrix with urea and sodium borate as additives. The particle showed increased degradation resistance by heat for 60 days, and UV radiation over 48h, with no more than 3% of degradation. This represents less than one tenth when compared to the UV degradation of commercial emulsifable concentrate. Even in those conditions, it was also able to promote controlled liberation of the active compound for up to 10 weeks de‐ pending on the formulation used.

In another work with acetamiprid, Takei *et al.* [17]produced microparticles with diameter of 30-150µm using poly-lactide (PLA) as the polymeric matrix. Initial results showed that mi‐ crospheres containing only PLA did not have a good release kinetic of the active chemical compound from its interior. It is presumably due to their tight structure and high hydropho‐ bicity, which hinders water diffusion and therefore limits the insecticide liberation. The in‐ clusion of poly(ε-caprolactone) (PCL) into the matrix in 50-80% weight were analyzed, with formation of microspheres of PLA/PCL blend with 20-120µm of the diameter, showing up to 88,5% of insecticide release in aqueous media over a 48h period.

30ºC, which is the melting temperature of the polymer,. Experiments were performed with Banded cucumber beetle *Diabrotica balteata* and Western corn rootworm *Diabrotica virgifera* as target insects at 20ºC and 32ºC, under and above the polymer melting point respectively. Mortality was compared to commercial granular formulation. At lower temperatures, the commercial formulation showed the best mortality. At higher temperatures the activity of the encapsulated formulation was better, showing about 90% of mortality for over 8 weeks. The commercial formulation had indeed lost some of its activity, presumably due to heat

Latheef *et al.* [13] tested several different polymers such as poly (methyl methacrylate) (PMMA), ethyl cellulose, poly(α-methylstyrene) and cellulose acetate butyrate to produce microcapsules of the insecticide sulprofos. Ethyl cellulose formulations were the only ones that had shown good results against eggs and larvae of the tobacco budworm *Heliothis vires‐ cens* in cotton plants. The results were comparable to the ones obtained with the use of an

In other to develop commercial formulation containing microencapsulated cyfluthrin, Ar‐ thur[14] evaluated its use against the rice weevil *Sitophilus oryzae* in stored wheat, for a peri‐ od of 8 months. Survival of beetles was statistically correlated with the concentration of the pyrethroid insecticide in the formulation. The average survival rate was only 12% when 4ppm was used, with constant activity throughout the entire experiment. This evidenced the

In the work carried out by Quaglia *et al.* [15], a hydrophobic waxy prepared through a mix‐ ture of di- and triglycerides of PEG esters was used to construct microspheres containing the insecticide carbaryl. Microparticles was obtained with particle size ranging from 16 to 20µm. Controllable release dynamics depended on the amount of gelucire used, Studies of release profiles from the encapsulated formulation showed a lower vertical mobility of the insecti‐ cide when compared to a commercial nonencapsulated formation. This suggested that the controlled release profile of the microcapsules may be useful to avoid or minimize ground‐

Cao *et al.* [16] produced diffusion-controlled microcapsules with diameter ranging from 2 to 20µm with encapsulated acetamiprid, an alkaline and high temperature-sensitive insecti‐ cide, using tapioca starch as matrix with urea and sodium borate as additives. The particle showed increased degradation resistance by heat for 60 days, and UV radiation over 48h, with no more than 3% of degradation. This represents less than one tenth when compared to the UV degradation of commercial emulsifable concentrate. Even in those conditions, it was also able to promote controlled liberation of the active compound for up to 10 weeks de‐

In another work with acetamiprid, Takei *et al.* [17]produced microparticles with diameter of 30-150µm using poly-lactide (PLA) as the polymeric matrix. Initial results showed that mi‐ crospheres containing only PLA did not have a good release kinetic of the active chemical compound from its interior. It is presumably due to their tight structure and high hydropho‐ bicity, which hinders water diffusion and therefore limits the insecticide liberation. The in‐

emulsifable-concentrate (EC) commercial formulation of sulprofos.

524 Insecticides - Development of Safer and More Effective Technologies

controlled release of the substance over a long period of time.

degradation.

water contamination.

pending on the formulation used.

In contrast to conventional desire to produce compounds with extended residual activity, quick-release microcapsules are demanded in certain areas of agriculture. However, some‐ times it is also necessary a quick liberation of the active compound from the matrix after the application. The strong backbone might pose as a problem to effectively deliver. Studies per‐ formed by Tsuda *et al.* [18,19] have shown that is possible to assemble "self-bursting" micro‐ capsules that retain its form in water suspension, but easily burst after solvent evaporation. They used the interfacial polymerization method to assemble spherical polyurethane micro‐ capsules containing the insecticide pyriprofixen, obtaining particles with mean diameter of 23µm. The entrapment ratio was 99% for all formulations tested, greatly improving the solu‐ bility of the pesticide in water. According to the results, there is a correlation between the wall thickness of the microcapsules and the self-bursting phenomenon. Tuning this property a controlled released can be achieved.

The effectiveness of encapsulated formulations, it is not restricted to extend the residual activi‐ ty of insecticides, but should also include the overcoming of problems associated with accu‐ mulation of recalcitrant organic pollutants that remains in ecosystems in amounts above the Maximum Residual Level (MRL). Therefore, it can be harmful to the environment and to peo‐ ple who might consume the treated crops. For instance, Guan *et al.* [20] encapsulated imidaclo‐ prid, a chloro-nicotinyl systemic and broad spectrum insecticide in a mixed sodium alginate/ chitosan microparticle through self-assembly layer-by-layer (LbL) methodology. The capsules showed a mean diameter of 7µm. Particles were impregnated with a photocatalyst made of SDS/TiO2/Ag, and the photocatalytic property and the insecticidal activity of the microcap‐ sule was evaluated. Prolonged residual activity of the encapsulated formulation was ob‐ served. The toxicity was higher in the *Martianus dermestoides* adult stage compared to the one of pure insecticide. In a field test with soybean [21], the nano-imidacloprid formulation prevent‐ ed the accumulation of the pesticide on the soybean leaves and soil. The results showed pro‐ nounced degradation over 25 days of trials when compared to commercial concentrate formulations, even though the initial concentration of both formulations was equivalent. In this way, regardless the initial effectiveness of the insecticide, safer levels of agrochemicals can be obtained in less time, improving the safety of insecticide application.

#### **2.3. Entomopathogenic microorganisms encapsulated**

Besides the chemical compounds, the micro- and nanotechnology have also been developed and applied to microorganisms that need special protection or to improve their solubility in aqueous phase. For instance, Ramírez-Lepe *et al.* [22] developed an aluminium-carboxyme‐ thylcellulose microcapsule with photoprotective agents for holding a *Bacillus thuringiensis* serovar *israelensis* (B.t.i.) spore-toxin complex named δ-endotoxin. The protein produced by this gram-positive bacterium during sporulation is extremely toxic to larval stage of some mosquitoes and flies which are vectors for important tropical diseases such as malaria and dengue. The encapsulated formulation was tested for its UV irradiation protective efficiency in laboratory conditions. While the protein in its natural form had lost all of its activity after 24 hours of exposure, encapsulated formulations showed up to 88% of larvae mortality.

released was proportional to the initial oil concentration on particles, with less than 20% of liberation for low oil concentrations, and about 60% liberation for high oil concentration.

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527

Yang *et al.* [32] assembled polyethylene glycol (PEG) nanoparticles loaded with garlic essen‐ tial oil using a melt-dispersion method, reaching over 80% of encapsulation efficiency, with round shaped nanoparticles of lower 240nm of average diameter. The encapsulated formu‐ lations had their insecticidal activity evaluated against adult red flour beetle *Tribolium casta‐ neum*. While the control experiment done with free garlic oil showed only 11% of efficiency over a five month period, the encapsulated formulation efficiency remained over 80% after five months. This was attributed to the slow and controlled release of the essential oil, and

The basic structure of the polymer chitosan was used by Lao *et al*. [33] to build the amphi‐ philic-modified *N*-(octadecanol-1-glycidyl ether)-*O*-sulfate chitosan (NOSCS). Octadecanol glycidyl ether and sulfate were the hydrophobic and the hydrophilic groups sources respec‐ tively. They successfully entrap the herbal insecticide rotenone in the polymer. This chemi‐ cal compound has been allowed for application in organic crop production due to its natural origin, short persistence in the environment, safety to non-target organisms and low resist‐ ance development. The encapsulation was necessary to defeat the problems of chemical sta‐ bility of the substance to environmental effects and also to improve the solubility of this pesticide in water, which is usually quite low (2.0x10-6g.L-1). Using the reverse micelle meth‐ od, the authors have assembled nanometric micelles with 167.7-214.0 nm of diameter, with values of critical micellar concentration (CMC) of those chitosan derivatives ranging from 3.55×10−3 to 5.50×10−3 g.L-1. Although the entrapment efficiency was not very high, they also improved the aqueous solubility of the chemical compound in 13,000 fold, up to 0.026g.L-1, favoring a controlled release of the substance in aqueous media. The complete controlled re‐ lease took more than 230 hours, almost 10 times more when compared to the chemical com‐

Chitosan derivatives were prepared [34]. They synthesized 6-*O*-carboxymethylated chitosan with anchorage of ricinoleic acid at the *N*-linkage, which further improve its solubility at neu‐ tral water (pH = 7.0), to encapsulate the herbal insecticide azadirachtin. Nanoparticles of 200-500nm were obtained by water dispersion with more than 50% of loading efficiency and tested for their stability in outdoor as controlled release systems. Results were compared against simple azadirachtin water dispersion and modified dispersion containing ricinoleic acid and azadirachtin. In 5 days of sun exposure, all content of control samples were lost, while the encapsulated formulation had a nearly constant residual concentration detected through‐ out the 12 days of the experiment, indicating that the nanoparticles produced were effective at controlling the degradation rate and the release mechanism of the botanical insecticide.

Extracts of Neem were prepared contend high concentration of azadirachtin being nanoen‐ capsulated by Forim *et al.* [35]. Through the use of poly-(ε-caprolactone) polymer, they pre‐ pared nanocapsules and nanospheres with average diameter of 150.0 and 250.0nm, respectively. The morphological analysis revealed spherical nanoparticles (Figure 1). The azadirachtin was used as reference. The nanoformulations showed high entrapment efficien‐

thus could be used as an effective pest control to stored products.

pound without nanoencapsulation.

In their turn, Tamez-Guerra *et al.* [23] also tested the encapsulation of the spore-toxin of *Ba‐ cillus thuringiensis* Berliner, evaluating over 80 formulations of spray-dried microcapsules made of lignin and corn flour with and without photoprotective agents. The best formula‐ tions showed improved insecticidal activity in laboratory tests against neonates of European corn borer *Ostrinia nubilalis* when compared to nonencapsulated or commercial formula‐ tions of the same endotoxin. In a field test, the microcapsules showed increased residual in‐ secticide activity in cabbage after 7 days against neonates of the cabbage looper *Trichoplusia ni* when compared to commercial formulations.

Very promising results have been obtained by the Agricultural Research Service of the US‐ DA regarding the encapsulation of biopesticides made of species-specific nucleopolyhedro‐ viruses (NPV) isolated from several insects, including celery looper *Anagrapha falcifera* (Tamez-Guerra *et al*., 2000 [24-26]), alfalfa looper *Autographa californica* [27], codling moth *Cydia pomonella* [28] and fall armyworm *Spodoptera frugiperda* [29]. In these works, formula‐ tions were developed using different mixtures of corn flour and lignin, through spray-dry‐ ing technique to encapsulate the viruses. All results obtained in laboratory and field tests performed have shown improvements in insecticidal activity, resistance to environmental conditions, like rain and UV light exposure, and a prolonged residual activity against pests in field studies. Samples were kept in storage for up to 12 months and maintained their in‐ secticidal activity.

#### **2.4. Novel micro and nanoparticles for bioinseticides**

Conventional protocols for encapsulation usually run under relatively high temperatures, which might be inadequate for preserving plant-derived essential oils integrity. Processes which use high pressure instead of temperature can be an alternative for encapsulating these sensible extracts. Varona *et al.* [30,31] developed new methods to produce stable parti‐ cles of lavandin (*Lavandula hybrida*) essential oil, using polyethylene glycol 9000 (PEG9000) or n-octenyl succinic (OSA) modified starches as the shell material. The methods for prepar‐ ing the microcapsules were based on PEG precipitation from a mixture of molten polymer and essential oil in supercritical CO2, and PGSS-drying an oil-in-water emulsion of the es‐ sential oil with OSA starch. The difference between these processes is the presence of water on the latter, which needs to be removed by carefully tuning the equipment conditions to promote water evaporation. Microcapsules produced by these methods show a mean parti‐ cle size of 10-500µm for PGSS, and 1-100µm for PGSS-drying. One important observation by scanning electron microscopy (SEM) images is that the experimental conditions can influ‐ ence the shape of the microparticles. While PEG particles were only spherical (the best shape for controlled release mechanism), in PGSS-drying needle-like structures are formed,, de‐ pending on the pre-expansion temperatures of the mixtures, The last one, probably does not hold the active ingredient, presenting some limitations to this specific method without fur‐ ther improvements. Release kinetics were evaluated over a 20-day period. The amount of oil released was proportional to the initial oil concentration on particles, with less than 20% of liberation for low oil concentrations, and about 60% liberation for high oil concentration.

in laboratory conditions. While the protein in its natural form had lost all of its activity after 24 hours of exposure, encapsulated formulations showed up to 88% of larvae mortality.

In their turn, Tamez-Guerra *et al.* [23] also tested the encapsulation of the spore-toxin of *Ba‐ cillus thuringiensis* Berliner, evaluating over 80 formulations of spray-dried microcapsules made of lignin and corn flour with and without photoprotective agents. The best formula‐ tions showed improved insecticidal activity in laboratory tests against neonates of European corn borer *Ostrinia nubilalis* when compared to nonencapsulated or commercial formula‐ tions of the same endotoxin. In a field test, the microcapsules showed increased residual in‐ secticide activity in cabbage after 7 days against neonates of the cabbage looper *Trichoplusia*

Very promising results have been obtained by the Agricultural Research Service of the US‐ DA regarding the encapsulation of biopesticides made of species-specific nucleopolyhedro‐ viruses (NPV) isolated from several insects, including celery looper *Anagrapha falcifera* (Tamez-Guerra *et al*., 2000 [24-26]), alfalfa looper *Autographa californica* [27], codling moth *Cydia pomonella* [28] and fall armyworm *Spodoptera frugiperda* [29]. In these works, formula‐ tions were developed using different mixtures of corn flour and lignin, through spray-dry‐ ing technique to encapsulate the viruses. All results obtained in laboratory and field tests performed have shown improvements in insecticidal activity, resistance to environmental conditions, like rain and UV light exposure, and a prolonged residual activity against pests in field studies. Samples were kept in storage for up to 12 months and maintained their in‐

Conventional protocols for encapsulation usually run under relatively high temperatures, which might be inadequate for preserving plant-derived essential oils integrity. Processes which use high pressure instead of temperature can be an alternative for encapsulating these sensible extracts. Varona *et al.* [30,31] developed new methods to produce stable parti‐ cles of lavandin (*Lavandula hybrida*) essential oil, using polyethylene glycol 9000 (PEG9000) or n-octenyl succinic (OSA) modified starches as the shell material. The methods for prepar‐ ing the microcapsules were based on PEG precipitation from a mixture of molten polymer and essential oil in supercritical CO2, and PGSS-drying an oil-in-water emulsion of the es‐ sential oil with OSA starch. The difference between these processes is the presence of water on the latter, which needs to be removed by carefully tuning the equipment conditions to promote water evaporation. Microcapsules produced by these methods show a mean parti‐ cle size of 10-500µm for PGSS, and 1-100µm for PGSS-drying. One important observation by scanning electron microscopy (SEM) images is that the experimental conditions can influ‐ ence the shape of the microparticles. While PEG particles were only spherical (the best shape for controlled release mechanism), in PGSS-drying needle-like structures are formed,, de‐ pending on the pre-expansion temperatures of the mixtures, The last one, probably does not hold the active ingredient, presenting some limitations to this specific method without fur‐ ther improvements. Release kinetics were evaluated over a 20-day period. The amount of oil

*ni* when compared to commercial formulations.

526 Insecticides - Development of Safer and More Effective Technologies

**2.4. Novel micro and nanoparticles for bioinseticides**

secticidal activity.

Yang *et al.* [32] assembled polyethylene glycol (PEG) nanoparticles loaded with garlic essen‐ tial oil using a melt-dispersion method, reaching over 80% of encapsulation efficiency, with round shaped nanoparticles of lower 240nm of average diameter. The encapsulated formu‐ lations had their insecticidal activity evaluated against adult red flour beetle *Tribolium casta‐ neum*. While the control experiment done with free garlic oil showed only 11% of efficiency over a five month period, the encapsulated formulation efficiency remained over 80% after five months. This was attributed to the slow and controlled release of the essential oil, and thus could be used as an effective pest control to stored products.

The basic structure of the polymer chitosan was used by Lao *et al*. [33] to build the amphi‐ philic-modified *N*-(octadecanol-1-glycidyl ether)-*O*-sulfate chitosan (NOSCS). Octadecanol glycidyl ether and sulfate were the hydrophobic and the hydrophilic groups sources respec‐ tively. They successfully entrap the herbal insecticide rotenone in the polymer. This chemi‐ cal compound has been allowed for application in organic crop production due to its natural origin, short persistence in the environment, safety to non-target organisms and low resist‐ ance development. The encapsulation was necessary to defeat the problems of chemical sta‐ bility of the substance to environmental effects and also to improve the solubility of this pesticide in water, which is usually quite low (2.0x10-6g.L-1). Using the reverse micelle meth‐ od, the authors have assembled nanometric micelles with 167.7-214.0 nm of diameter, with values of critical micellar concentration (CMC) of those chitosan derivatives ranging from 3.55×10−3 to 5.50×10−3 g.L-1. Although the entrapment efficiency was not very high, they also improved the aqueous solubility of the chemical compound in 13,000 fold, up to 0.026g.L-1, favoring a controlled release of the substance in aqueous media. The complete controlled re‐ lease took more than 230 hours, almost 10 times more when compared to the chemical com‐ pound without nanoencapsulation.

Chitosan derivatives were prepared [34]. They synthesized 6-*O*-carboxymethylated chitosan with anchorage of ricinoleic acid at the *N*-linkage, which further improve its solubility at neu‐ tral water (pH = 7.0), to encapsulate the herbal insecticide azadirachtin. Nanoparticles of 200-500nm were obtained by water dispersion with more than 50% of loading efficiency and tested for their stability in outdoor as controlled release systems. Results were compared against simple azadirachtin water dispersion and modified dispersion containing ricinoleic acid and azadirachtin. In 5 days of sun exposure, all content of control samples were lost, while the encapsulated formulation had a nearly constant residual concentration detected through‐ out the 12 days of the experiment, indicating that the nanoparticles produced were effective at controlling the degradation rate and the release mechanism of the botanical insecticide.

Extracts of Neem were prepared contend high concentration of azadirachtin being nanoen‐ capsulated by Forim *et al.* [35]. Through the use of poly-(ε-caprolactone) polymer, they pre‐ pared nanocapsules and nanospheres with average diameter of 150.0 and 250.0nm, respectively. The morphological analysis revealed spherical nanoparticles (Figure 1). The azadirachtin was used as reference. The nanoformulations showed high entrapment efficien‐ cy (> 95%) for this compound and a UV stability at least of 30 times more when compared with commercial products.

ruling the (bio) availability of the active compound after the application [48]. They find the greatest applicabilities in two major agricultural fields: nutrition and protection. In the first one, CRFs are employed in the delivery of fertilizers [49-51]. In the second one, CRFs are mostly used to target plague insects in a sustainable way [52,53], but they can also be ap‐ plied to block the growth of weeds [54]. Tomioka *et al.*, 2010. Controlled release formula‐ tions become especially interesting in cases of antagonist activity of biocides, what can naturally leads to a lower in effectiveness of one or both compounds. In this case the formu‐ lation should be "programmed" to release each one at different times [55,56]. Furthermore, still talking about protection, the application of CRFs in wood surfaces, like furniture or floor covering, helps to prevent the deterioration. Van Voris *et al.* [57] patented a formula‐ tion in which an insecticide is continually released in a minimum level for a long period of time and is absorbed by the wood. It thus creates a "chemical barrier", blocking the insect

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Most of those controlled release biocides applications were and still are successfully made

Micro- and nanomaterials-based formulations are known for some decades. The first micro‐ capsule-based formulation became commercially available in the 1970s [58]. Nanocapsules have been widely used in medicinal area as drug carrier in treatment of diverse diseases

Microencapsulation has been used as a versatile tool for hydrophobic pesticides, enhancing their dispersion in aqueous media and allowing a controlled release of the active compound. The use of nanotechnology is a recent approach, and has been a growing subject on several different areas of the science, with an overwhelming perspective. In general, materials that are assembled in nanometric scales (<1000nm) have distinct and almost always better char‐ acteristics when compared to the same material built in a conventional manner [62]. One

rials in nanometric scale may be controlled to promote an efficient assemble of a structure which could present several advantages, such as the possibility to better interaction and mode of action at a target site of the plant or in a desired pest due to its tunable controlled release system and larger superficial area, acting as an artificial immune system for plants [34,63]. As smart delivery systems, they confer more selectivity, without hindering in the bi‐ oactive compounds towards the target pathogen [65]. Other advantages of the use of nano‐ particle insecticides are the possibility of preparing formulations which contain insoluble compounds that can be more readily dispersed in solution. It reduces the problems associat‐ ed with drifting and leaching, due to its solid nature, and leads to a more effective interac‐ tion with the target insect. These features enable the use of smaller amount of active compound per area, as long as the formulation may provide an optimal concentration deliv‐ ery for the target insecticide for longer times. Since there is no need for re-applications, they also decrease the costs), reduce the irritation of the human mucous-membrane, the phyto‐ toxicity, and the environmental damage to other untargeted organisms and even the crops themselves [65,66]. In a few words, nanotechnology can be applied in several ways in order

m). In general, the chemical properties of mate‐

attacks.

due to the advances in nanotechnology area.

[59], from tropical ones [60] up to cancer [61].

nanometer is a billionth of a meter (1nm = 109

to enhance efficacy of insecticides in crops.

**Figure 1.** Scanning electron microscopy images of nanoparticles containing extracts of Neem.

#### **2.5. Commercial products**

The interesting results obtained in academic researches over the last few decades have been closely followed by several companies. Nevertheless, R&D in nano-based agrochemicals is led mainly by world's largest agroscience companies, further enhancing their market share and consolidating the market structure based on oligopoly that have been seen in late 20th century and early 21th century, when the 10 biggest companies hold around 80% of market [36].

Some companies over the last decade, such as Syngenta, Bayer, Monsanto, Sumitomo, BASF, and Dow Agrosciences have already deposited several different patents comprising a wide range of protocols for production and application of encapsulated formulations, which can be used to produce nanoinsecticides [37-46]. Despite the hard work and heavy investment, no commercial nano-insecticide formulation has been extensively commercialized up to 2012.

Along with those big industries, several other companies, as well as individual researches have been actively depositing patents in the area, thus promoting even more the research and investments in this new field of applied technology. However, as strongly reinforced throughout the world by dozens of organizations such as the ETC Group, the impact of nanotechnology is still unclear, and care should be taken to assure that its use will not bring more problems than solutions [47].


**8. Conclusions**

For years, zeolite crystals have been used in various technological fields of great industrial interest such as catalysts, ion exchangers, and adsorbents in chromatographic applications. Today, the preparation of crystalline zeolite membranes plays a central role in many new applicative fields such as catalytic zeolite membrane reactors, gas separations, drug delivery, anticancer activity modulation, food toxicology, enzyme/protein adsorption, and antimicrobial nanotechnologies. Zeolite crystals and membranes are key materials for selective adsorption of biological molecules, drugs, and chemotherapy drugs. Moreover, the zeolite membranes represent a synthetic scaffold suitable, ideal, and able to guarantee the survival, growth, and correct differentiation of human normal and cancer cells. The development of zeolite membranes with versatile physics-chemical properties may constitute the goal for new components in biomedical and biotechnological applications.


*3.4. Mac-1 and TLR2 Surface Neutrophil Receptors Are Involved in Farnesol-Induced Activation of Netosis*

Many of the neutrophil surface receptors are involved in the recognition of molecules derived from pathogenic microorganisms, which leads to activation of the netosis signaling pathway and the NET release. We selected six receptors known to be able to mediate the release of NETs and checked their role in FOH-induced netosis. The receptors were blocked with specific antibodies, and the neutrophils were stimulated with selected concentrations of FOH for three hours. A decrease in the level of fluorescence of Sytox Green-stained DNA suggests the role of this receptor in the activation of netosis. Among selected receptors, we identified that the inhibition of CD11b and CD18 caused a ca. 2.5-fold decrease in the level of released NETs (Figure 6). This result strongly suggests the important role of these receptors in the recognition of FOH and activation of netosis. Moreover, inhibition of the TLR2 receptor caused a reduction of the level of NETs to about 50% of PMA-treated response, also indicating the participation of this receptor in netosis. A small contribution to the activation of

FOH-induced netosis could also be assigned to CD11a receptor whose blockade caused a 20% decrease in the amount of released DNA. Other receptors tested—CD16 and TLR4—did not seem to be essential for FOH-induced netosis.

**Figure 6.** The participation of selected neutrophil receptors in FOH-activated netosis. Neutrophils (2.2 × 105) were preincubated with antibodies (ab, 1 μg/mL) against the selected neutrophil receptors and then netosis was induced for 3 h by 25 nM PMA, 100 μM FOH, and 200 μM FOH. IgG antibody was used as an isotype control. Released NETs were digested with MNase, and collected supernatants were stained with Sytox Green, followed by fluorescence measurements. The results are the means of two replicates ± S.E.M., represented as a percentage relative to the PMA-treated control. ANOVA and Dunnett's post-tests were used. Asterisks denote statistical significance (*p* > 0.1234 ns, \*\* *p* ≤ 0.0021, \*\*\* *p* ≤ 0.0002, \*\*\*\* *p* < 0.0001).

#### *3.5. Farnesol Leads to the Activation of Protein Kinase C and ERK1*/*2*

PKC plays a crucial role in the netosis signal pathway. We checked the level of PKC activation in lysates of neutrophils previously treated with two concentrations of FOH or FA for one hour. After pre-purification of the lysates on DEAE cellulose, the kinase activity was determined based on the phosphorylation of the synthetic peptide, which was then subjected to electrophoretic separation. Figure 7a presents an electrophoretic separation of a synthetic peptide whose amount in the phosphorylated form determines the level of PKC activation. Figure 7b shows quantitative PKC activity in assayed samples corresponding to the densitometrically analyzed bands of the phosphorylated form of the peptide on an electrophoretic gel.

The results showed that PKC activity in neutrophils treated with FOH increased rapidly, reaching a two-fold higher level than in unstimulated cells, comparable to that obtained for chemically induced (PMA) activation of this enzyme. The PKC activity leads to the activation of subsequent netosis mediators. On the other hand, PMNs treated with FA did not show any changes in PKC activity. The lack of PKC activation by FA confirms that neutrophils do not release NETs in the presence of this QSM.

**Figure 7.** Activation of Protein Kinase C (PKC) in QSM–treated neutrophils. Neutrophils (5 × 105) were stimulated with selected concentrations of FOH, FA, and 25 nM PMA for 1 h. Then, cell lysate was added to the PKC substrate solution-PepTag® C1 Peptide, and after 30 min separated electrophoretically on an agarose gel. Unstimulated cells were negative cellular control, and PMA-treated cells were positive cellular control. Purified PKC enzyme was used for an assay positive control (control of C1 Peptide phosphorylation), negative control was C1 peptide without any PKC enzyme. (**a**) PKC activation as a percentage of phosphorylated C1 peptide, a substrate for PKC. The values represent the densitometrically-analyzed bands on the electrophoretic gel, assigned as "Active" in (**b**).

The results of the analysis of ERK1/2 kinase activation in neutrophils treated with FOH are shown on Figure 8. The activation of ERK1/2 by 100 μM FOH is two-fold higher than in unstimulated cells, but 200 μM FOH caused three-fold greater phosphorylation of this enzyme. These findings suggest that the activation of the signaling pathway by FOH in neutrophils involving ERK1/2 in a dose-dependent manner.

**Figure 8.** Activation of ERK1/2 in FOH-treated neutrophils. Neutrophil cells (1 × 106 cells/well) were suspended in PBS in 12-well microplate and stimulated with FOH at concentrations of 100 μM and 200 μM. Unstimulated cells served as a negative control, and PMA-treated cells were a positive control. After 1 h, the cells were lysed, and the amount of total and phosphorylated ERK1/2 was quantified using ELISA Kit SimpleStep (Abcam). Data represent the mean values of absorbance ± S.E.M. from two replicates as the percentage ratio of phosphorylated ERK1/2 to total ERK1/2. ANOVA and Tukey post-tests were used. Asterisks denote statistical significance (*p* > 0.1234–ns, \* *p* ≤ 0.0332, \*\* *p* ≤ 0.0021).

#### *3.6. The ROS-Dependent Netosis Pathway is Activated by Farnesol*

Stimulation of surface receptors involved in the induction of netosis leads to the activation of selected mediators of the signaling pathway. We checked the role of the five primary mediators involved in ROS-dependent and ROS-independent netosis pathways. For this purpose, selected mediators were blocked in PMNs using specific inhibitors before neutrophil activation with 200 μM FOH. Fluorescence of Sytox Green-stained extracellular DNA was used to determine NET release.

The results (Figure 9) indicate that each of the tested mediators was involved in FOH-induced netosis, but with a relative variable contribution. Syk and Src kinases co-operated with neutrophil surface receptors, and their involvement in netosis activation appeared to be significant. The inhibition of these proteins caused a 50% decrease in the amount of released NETs. In turn, the role of PI3K in FOH-induced netosis seemed to be less important. PI3K probably played a significant role in the ROS-independent netosis signaling pathway. In our experiments, blockade of this kinase caused a decrease in the amount of extracellular DNA by 20% relative to the control.

**Figure 9.** Role of selected signal mediators in activation of netosis by FOH. Neutrophils (2.2 × 105) were preincubated with inhibitors of the indicated signaling mediators: Syk—30 μM piceatannol, Src—10 μM PP2, PI3K—25 μM LY29004, ERK1/2—10 μM U0126, NADPH oxidase—5 μM DPI. Cells were then incubated with FOH at 200 μM concentration for 3 h to induce netosis. Neutrophils not treated with inhibitors but stimulated with FOH served as a control. The data are presented as means ± S.E.M. from two replicates and are expressed as a percentage ratio relative to the control. ANOVA and Tukey post-tests were used. Asterisks denote statistical significance (*p* > 0.1234 ns, \* *p* ≤ 0.0332, \*\* *p* ≤ 0.0021, \*\*\* *p* ≤ 0.0002).

ERK1/2 and NADPH oxidase also participate in the activation of the ROS-dependent netosis pathway. The release of NETs depending on ROS production appears to be the primary netosis mechanism involved in neutrophil responses to FOH, as the FOH stimulation of PMNs with blocked ERK1/2 resulted in a 50% reduction of the amount of released NETs. Moreover, NADPH oxidase, which is responsible for the production of ROS in the cells, seems to play the most significant role in the netosis process induced with FOH. Only 20% of DNA was released by neutrophils with the blocked activity of NADPH oxidase compared to the control cells. This result and data showing increased ROS production by neutrophils treated with farnesol confirm that FOH-induced netosis is a ROS-dependent process.

#### *3.7. Farnesol Is a Chemoattractant for Neutrophils*

Additionally, we showed that FOH may be a chemoattractant for PMNs. To support this hypothesis, the neutrophils were placed in chambers to measure chemotactic ability, and then the chambers were transferred to the FOH solutions at the concentration within a range of 50 to 400 μM. As the negative control, PBS was used, whereas the positive control was represented by the responses of neutrophils to 1 μM solution of fMLP. The number of cells that passed through the membrane according to the factor gradient was counted after one hour of treatment. The obtained results (Figure 10) showed that FOH is recognized by neutrophils as a chemoattractant, causing the PMN movement toward the concentration gradient. The number of cells that passed through the membrane was proportional to FOH concentration in the range of 50 to 200 μM, that corresponds to the concentration of FOH detected under in vivo conditions [23]. The higher concentrations of FOH did not cause any further increase in neutrophil migration, but anyway the maximal level of neutrophil chemotaxis upon FOH treatment reached 60% of the response to fMLP, the positive control. These results suggest that neutrophils can migrate to the *Candida* infection sites in response to QSMs released by the yeast.

**Figure 10.** Chemotactic activity of FOH-treated neutrophils. CellTracker Red labeled neutrophil cells (1 × 106 cells/well) were placed into chemotaxis chambers being in contact with different concentration of FOH or 1 μM fML (a positive control). Negative control was PBS. After 1 h of incubation, the chambers were removed, and migrated cells were counted. Data represent the mean number of cells ± S.E.M. from three replicates. ANOVA and Dunnett's multiple comparisons post-tests were used. Asterisks denote statistical significance (*p* > 0.1234 ns, \*\*\*\* *p* < 0.0001).

#### **4. Discussion**

Quorum sensing molecules are essential virulence factors of many pathogenic microorganisms-bacteria as well as fungi. The release of QSMs ensures the fast local communication between cells in the infected area, growth synchronization, as well as response to environmental changes of temperature and pH, the presence of biocidal compounds, etc. [34,35].

*C. albicans* yeast mainly use QSMs to regulate growth, change of morphological form and also create biofilms [36]. However, these molecules have an impact on many host cells, like immune cells, such as macrophages, DCs, and neutrophils [20,23].

Our results presented in this paper indicated an important role of neutrophils in the response to QSMs produced by *C. albicans*, which is based on the release of NETs. We showed that farnesol, one of the three compounds identified as *C. albicans* QSMs (FOH, FA and TR) was responsible for activating the netosis, a finding that had not been presented in the literature previously.

The role of FOH released by *C. albicans*is still not fully understood, but it is known that the presence of this compound inhibits the growth of fungal biofilm, and also blocks the change in the morphological transition of fungal cells from blastospores to filamentous forms. Also, high concentrations of FOH can stimulate the yeast to reverse switch from invasive filamentous hyphae back to blastospore [37]. As we presented in the current work, FOH produced at different local concentrations at the site of infection caused the neutrophil response involving NET release. The finding is important for the host defense against *C. albicans* cells because current knowledge has suggested that the morphological form of the fungus determines the mechanism of neutrophil response [28,30]. Some findings indicated that the size of the fungal cells determines the release of NETs, suggesting that the large, filamentous form of *C. albicans* is responsible for activating the netosis [28]. Other studies indicated that the composition of the cell wall of the filamentous form, as well as the released proteolytic enzymes are crucial for the stimulation of neutrophils to NET production [30,38]. Regardless of the identified NET triggers, all studies pointed that *C. albicans* blastospores generate much smaller amounts of NETs, suggesting that this morphological form of *C. albicans* cells can "hide" from neutrophils and avoid the killing. Perhaps, fungi use FOH to inhibit their cell filamentation and progress of infection, just to survive in milieu

infiltrated by neutrophils This situation is dangerous for the host because the 'invisible' to neutrophils intruder cells can survive and under favorable conditions, develop the difficult to treat, secondary infections [39]. Therefore, the ability of neutrophils to recognize FOH, the essential QSM molecule of the fungus, and release NETs in the response to its production at the place of infection, can significantly affect the effectiveness of host defense against *C. albicans* presence. We confirmed the recognition of FOH by neutrophils using chemotaxis analysis, showing that FOH is an efficient chemotactic factor for neutrophils. To date, there is no literature data showing neutrophil migration caused by FOH. However, FOH is known to be a chemoattractant for macrophages [40].

Moreover, given that the microbiocidal activity of NETs covers a certain area of infection, a direct identification of fungal cells by neutrophils is not required, and the presence of FOH may be sufficient.

Studies performed by Leonhardt et al. regarding the effect of FOH on neutrophils did not show any increasing ability of these host cells to phagocyte and kill *C. albicans* cells. However, the viability of *C. albicans* was analyzed after 1 h of contact, while the classic ROS-dependent netosis pathway leads to NET release after about 2–3 h of neutrophil activation [23]. The short time period used in Leonhard's analysis may explain the lack of changes in the fungal cell vitality. The same studies showed an increased FOH concentration-dependent release of elastase, myeloperoxidase, and lactoferrin–granule proteins identified in NETs [23,29]. Thus, our observations of NET release may explain the presence of granule proteins upon neutrophil treatment with FOH in Leonhardt's studies.

Activation of netosis by QSMs has been described in only one case—the *Pseudomonas aeruginosa* bacterium. It was shown that mutations in the quorum-sensing regulatory gene lasR affect the amount and structure of released NETs. In addition, stimulation of neutrophils with purified protein, recombinant endotoxin-free LasA induced NETs in a concentration-dependent manner [41].

The other two analyzed QSMs-FA and TR–did not activate netosis. There are also no literature reports on the impact of these compounds on the functioning of neutrophils. Only TR was identified as a protective agent against phagocytic killing by neutrophils, however, the mechanism of its action is still unknown. One study showed the antioxidant activity of TR [42], which could influence the neutrophil response by blocking ROS production, required for proper functioning of netosis mechanisms. However, other studies showed no antioxidant activity of TR, thus leaving the mechanism of its action on neutrophils unrecognized [18].

The production of extracellular DNA but not apoptosis was demonstrated in this work for neutrophils treated with FOH at the range of concentrations observed locally at the site of *C. albicans* infection. This result is consistent with the observation of Leonhardt et al. [23]. In contrary, we presented that FA is active inducer of neutrophil apoptosis in a dose-dependent manner, the finding that can explain the lack of netosis in neutrophil response to this QSM. These results were confirmed by the cytometric analysis showing the morphological changes of neutrophil cells observed during the FOH-induced netosis process but absent upon treatment with FA.

During the contact of *C. albicans* cells with neutrophils, resulting in NET production, the more common ROS-dependent mechanism of netosis was adopted. The analysis of ROS production by PMNs stimulated with FOH showed that neutrophils release ROS in just a few minutes after activation confirming the ROS-dependent netosis pathway used by neutrophils in response to FOH. In addition, Gilbert et al., identified farnesyl thiotriazole (FTT), a FOH precursor as an activator of NADPH oxidase in neutrophils [43]. However, the production of ROS in response to FA and TR was not observed, confirming a lack of response in the netosis way.

Knowing that FOH is a chemoattractant for neutrophils, and also activates the ROS-dependent netosis pathway, the role of selected surface receptors in activation of netosis by this QSM was analyzed. The results pointed the CD11b/CD18 and TLR2 receptors as being involved in the recognition of FOH and further activation of neutrophils. The CD11b/CD18 receptor engagement in neutrophil chemotaxis and netosis responses to fungal infection was previously demonstrated [30]. Also Leonhardt et al. observed that during stimulation of PMNs by FOH, the level of CD11b receptor on the cell surface increased, while the amount of CD16 receptor decreased [23]. However, Ghosh et al. showed increased

TLR2 and Dectin-1 expression in FOH-treated macrophages [22]. No analogous analysis was performed for neutrophils, however, the effect of FOH on TLR2 may also be significant in PMNs.

Further, the role of known mediators of netosis signaling pathway was verified upon neutrophil treatment with FOH. We showed FOH-induced phosphorylation of PKC, an important signal transducer of netosis. Activation of PKC results in the activation of NADPH oxidase and the production of significant amounts of ROS [44]. This result confirms and explains the role of ROS produced by neutrophils in contact with FOH. There is no literature data on the activation of PKC by FOH in neutrophils; however, in human acute leukemia CEM-C1 cells, the synthesis of diacylglycerol (DAG), a PKC activator, was observed in response to 20 μM FOH [45]. In addition, FTT seems to be also a PKC activator [43].

Inhibition of netosis pathway mediators such as Syk, Src, PI3K, and ERK1/2 caused a decrease in the amount of released NETs. Previously, only the pro-apoptotic activity of FOH was known, leading to reduction of PI3K expression in cells and ERK1/2 in HeLa and DU145 prostate cancer cells [46,47]. In turn, Joo et al. also pointed to the pro-apoptotic effect of FOH on cells, however, by activation of ERK1/2 [48]. However, our results did not show the inhibition of these mediators. In contrary, ERK1/2 kinase activity showed a two-fold increase at the activation of the netosis mechanism during which the apoptosis pathway in the PMNs was blocked.

Our results showed for the first time the role of FOH in the neutrophil recognition of fungal infection at an early stage of microbial invasion. It seems that the regulation of fungal cell morphology by FOH during the progress of fungal infection can also be sensed by the host. Neutrophils, the cells of the first line of host defense can "eavesdrop" the fungal cell communication that uses the quorum sensing molecule and then quickly migrate to the pathogen's habitat and kill or limit its spread by using NETs. The discovery of QSMs that can activate netosis can be of great importance for the developing of effective therapy against early candidiasis.

**Author Contributions:** Conceptualization, M.R.-K. and M.Z.; methodology, M.R.-K. and M.Z.; conceived and designed the experiments, M.R.-K. and M.Z.; all experiments performed by M.Z., except the data shown in Figure 1b, prepared by K.W.; writing—original draft preparation, M.R.-K. and M.Z.; writing—review and editing, M.R.-K. and M.Z.; funding acquisition M.R.-K.

**Funding:** This work was funded by a grant from the National Science Centre of Poland (grant number 2015/17/B/NZ6/02078 to MRK).

**Acknowledgments:** Thanks to Andrzej Kozik for critical discussion during the manuscript preparation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Perspective*


*Article* **Ecological Momentary Assessment to Obtain Signal Processing Technology Preference in Cochlear Implant Users**

**Matthias Hey 1,\*, Adam A. Hersbach 2, Thomas Hocke 3, Stefan J. Mauger 4, Britta Böhnke 1 and Alexander Mewes 1**

**Abstract:** Background: To assess the performance of cochlear implant users, speech comprehension benefits are generally measured in controlled sound room environments of the laboratory. For fieldbased assessment of preference, questionnaires are generally used. Since questionnaires are typically administered at the end of an experimental period, they can be inaccurate due to retrospective recall. An alternative known as ecological momentary assessment (EMA) has begun to be used for clinical research. The objective of this study was to determine the feasibility of using EMA to obtain in-themoment responses from cochlear implant users describing their technology preference in specific acoustic listening situations. Methods: Over a two-week period, eleven adult cochlear implant users compared two listening programs containing different sound processing technologies during everyday take-home use. Their task was to compare and vote for their preferred program. Results: A total of 205 votes were collected from acoustic environments that were classified into six listening scenes. The analysis yielded different patterns of voting among the subjects. Two subjects had a consistent preference for one sound processing technology across all acoustic scenes, three subjects changed their preference based on the acoustic scene, and six subjects had no conclusive preference for either technology. Conclusion: Results show that EMA is suitable for quantifying real-world selfreported preference, showing inter-subject variability in different listening environments. However, there is uncertainty that patients will not provide sufficient spontaneous feedback. One improvement for future research is a participant forced prompt to improve response rates.

**Keywords:** cochlear implant; signal processing; hearing in noise; EMA; ecological momentary assessment; acoustic environment; BEAM; ForwardFocus


**4. Detection of POPs at trace level in real environmental samples**

In sections 3 we introduced the SERS method to detect trace amount PCBs. In those experi‐ ments, the PCBs are in acetone solutions, as fundamental study. In this section we introduce some examples in practical trace POPs detection.

ized away. Then, we found PCBs Raman signal with the SERS method described before. Figs 14 (a) and (b) show the SERS spectra of pure white spirit and white spirit with 10-4mol/L PCBs, respectively. One can recognize characteristic Raman peaks of PCBs around 1590,

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**Figure 13.** SERS spectra of PCBs in dry soil samples after being treated by acetone: (a) ~10-5mol/L; (b) ~10-6mol/L; (c)

1290, 1240, 1030 and 1000 cm-1 in Fig 14 (b).

~10-7mol/L; (d) ~10-8mol/L.

#### **4.1. Detection of PCBs in dry soil samples**

The polluted soil samples were dried and made into small powers which were acquired from the Nanjing Institute of Soil (China). With a combination of the high-resolution gas chromatography and mass spectrometry techniques, sample I proved to contain about 5 μg/g PCBs and sample II proved to contain about 300 μg/g PCBs. 0.2 g soil sample I was put into 20 mL acetone and was agitated uniformly for about 5 minutes. This suspension was precipitated for 30 minutes and the transparent acetone solution in the upper layer was taken as solution sample A. 0.2 g soil sample I was put into 200 mL acetone and sol‐ ution sample B was obtained through the aforementioned process. 0.2 g soil sample II was put into 20 mL acetone to obtain solution sample C and was put into 200 mL ace‐ tone to obtain solution sample D.

The Ag nanorods SERS substrates were put into the solution samples A, B, C and D, respec‐ tively. After 30 minutes, the Ag nanorods substrates were taken out of the solutions and the acetone on the substrates was blown away using a nitrogen flow. The Raman spectra of these substrates dipped into solution samples were measured by a Renishaw Raman 100 spectrometer using a 633 nm He-Ne laser as the excitation source at room temperature.

Figs 13(a), (b), (c) and (d) show the measured Raman spectrum of the Ag substrates dipped into sample A, B, C and D, respectively. From Figs 13 (a), (b) and (c), one sees peaks at ~1600, 1280, 1240, 1150, 1030 and 1000 cm-1 clearly, demonstrating the common feature of PCBs. The peaks around 1590~1600cm-1 present benzene stretching vibration mode; the peak around 1280cm-1 presents CC bridge stretching vibration mode; the peak around 1030cm-1 presents CH bending in-plane mode; the peak around 1000cm-1 presents trigonal breathing vibration mode; and peaks around 1240~1250cm-1 and 1140~1200cm-1 present the vibration peaks induced by Cl substituent. These characteristic peaks suggest that PCBs in dry soil can be detected by the SERS method by dissolving into acetone. The most widely used PCBs are trichlorobiphenyls and pentachlorobiphenyls, we assumed that the molecular weight of the PCBs in the soil samples is 300, then the concentration of the PCBs acetone solution in solu‐ tion sample A, B, C and D are about 10-5mol/L, 10-5mol/L, 10-6mol/L, 10-7mol/L, and 10-8mol/L, respectively. Thus, with silver nanorod substrates, 5ug/g PCBs in dry soil samples can be detected by the SERS method.

#### **4.2. Detection of PCBs in white spirit**

PCBs in white spirit can also be detected by the SERS method with silver nanorod sub‐ strates. The concentration of PCBs in white spirit is about 10-4mol/L. We put a drop of PCBs "polluted" white spirit on the silver nanorod substrates and made the white spirit volatil‐ ized away. Then, we found PCBs Raman signal with the SERS method described before. Figs 14 (a) and (b) show the SERS spectra of pure white spirit and white spirit with 10-4mol/L PCBs, respectively. One can recognize characteristic Raman peaks of PCBs around 1590, 1290, 1240, 1030 and 1000 cm-1 in Fig 14 (b).

**4. Detection of POPs at trace level in real environmental samples**

some examples in practical trace POPs detection.

**4.1. Detection of PCBs in dry soil samples**

48 Organic Pollutants - Monitoring, Risk and Treatment

tone to obtain solution sample D.

can be detected by the SERS method.

**4.2. Detection of PCBs in white spirit**

In sections 3 we introduced the SERS method to detect trace amount PCBs. In those experi‐ ments, the PCBs are in acetone solutions, as fundamental study. In this section we introduce

The polluted soil samples were dried and made into small powers which were acquired from the Nanjing Institute of Soil (China). With a combination of the high-resolution gas chromatography and mass spectrometry techniques, sample I proved to contain about 5 μg/g PCBs and sample II proved to contain about 300 μg/g PCBs. 0.2 g soil sample I was put into 20 mL acetone and was agitated uniformly for about 5 minutes. This suspension was precipitated for 30 minutes and the transparent acetone solution in the upper layer was taken as solution sample A. 0.2 g soil sample I was put into 200 mL acetone and sol‐ ution sample B was obtained through the aforementioned process. 0.2 g soil sample II was put into 20 mL acetone to obtain solution sample C and was put into 200 mL ace‐

The Ag nanorods SERS substrates were put into the solution samples A, B, C and D, respec‐ tively. After 30 minutes, the Ag nanorods substrates were taken out of the solutions and the acetone on the substrates was blown away using a nitrogen flow. The Raman spectra of these substrates dipped into solution samples were measured by a Renishaw Raman 100 spectrometer using a 633 nm He-Ne laser as the excitation source at room temperature.

Figs 13(a), (b), (c) and (d) show the measured Raman spectrum of the Ag substrates dipped into sample A, B, C and D, respectively. From Figs 13 (a), (b) and (c), one sees peaks at ~1600, 1280, 1240, 1150, 1030 and 1000 cm-1 clearly, demonstrating the common feature of PCBs. The peaks around 1590~1600cm-1 present benzene stretching vibration mode; the peak around 1280cm-1 presents CC bridge stretching vibration mode; the peak around 1030cm-1 presents CH bending in-plane mode; the peak around 1000cm-1 presents trigonal breathing vibration mode; and peaks around 1240~1250cm-1 and 1140~1200cm-1 present the vibration peaks induced by Cl substituent. These characteristic peaks suggest that PCBs in dry soil can be detected by the SERS method by dissolving into acetone. The most widely used PCBs are trichlorobiphenyls and pentachlorobiphenyls, we assumed that the molecular weight of the PCBs in the soil samples is 300, then the concentration of the PCBs acetone solution in solu‐ tion sample A, B, C and D are about 10-5mol/L, 10-5mol/L, 10-6mol/L, 10-7mol/L, and 10-8mol/L, respectively. Thus, with silver nanorod substrates, 5ug/g PCBs in dry soil samples

PCBs in white spirit can also be detected by the SERS method with silver nanorod sub‐ strates. The concentration of PCBs in white spirit is about 10-4mol/L. We put a drop of PCBs "polluted" white spirit on the silver nanorod substrates and made the white spirit volatil‐

**Figure 13.** SERS spectra of PCBs in dry soil samples after being treated by acetone: (a) ~10-5mol/L; (b) ~10-6mol/L; (c) ~10-7mol/L; (d) ~10-8mol/L.

**5. Summary**

**Author details**

Zhengjun Zhang1

**References**

detection via silver nanostructure.

hua University, Beijing, P. R. China

41(6): 589–624.

lyticaChimicaActa, 2005, 531(2): 249–256.

, Qin Zhou1,2 and Xian Zhang1

\*Address all correspondence to<EMAIL_ADDRESS>
In this chapter, it is introduced that although persistent organic pollutants such as PCBs are difficult to detect at trace amount, they can be detected and recognized rapidly via the SERS technique. Ag nanostructured SERS substrates prepared by the glancing angle deposition method are excellent at detection and their sensitivity can be further improved by tuning the thin underlayer films. With well designed and prepared Ag nanostructured SERS sub‐ strates, pentachlorinated biphenyl molecules are detected and recognized at trace level us‐ ing the SERS method. These series of studies provide a potential method for trace pollutant

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1 Advanced Materials Laboratory, Department of Materials Science and Engineering, Tsing‐

2 Institute of nuclear and new energy technology, Tsinghua University, Beijing, P. R. China

[1] Ross G. The public health implications of polychlorinated biphenyls (pcbs) in the en‐

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**Figure 14.** SERS spectra of white spirit without and with PCBs.

#### **4.3. Detection of Melamine in milk**

In the year 2009, milk produced by Sanlu Co. (China) was found to contain amounts of Mel‐ amine in much higher concentrations than usual. Milk with Melamine seems to contain more protein when detecting nitrogen concentration, but it is poisonous to children. With the SERS method with silver nanorods as substrates, we detected Melamine in milk. Figs 15 (a) and (b) show the SERS spectra of pure Melamine and milk with Melamine.

**Figure 15.** SERS spectra of Melamine and trace Melamine in milk.